High concentration antibody formulations

ABSTRACT

The present disclosure relates to, inter alia, stable aqueous solutions comprising a high concentration of an antibody that binds to human complement component C5 and methods for preparing the solutions. The disclosure also provides methods for treating or preventing complement-associated disorders (for example, age-related macular degeneration or rheumatoid arthritis) using the solutions. Also featured are therapeutic kits containing one or more of the solutions and a means for administering the solutions to a patient in need such a treatment.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/413,268, filed Mar. 6, 2012, which is a continuation-in-part of U.S.application Ser. No. 11/127,438, filed May 11, 2005, which is acontinuation-in-part of U.S. application Ser. No. 10/655,861, filed Sep.5, 2003, which claims the benefit of priority to U.S. ProvisionalApplication No. 60/469,189, filed May 9, 2003 and U.S. ProvisionalApplication No. 60/408,571, filed Sep. 6, 2002. application Ser. No.13/413,268 also claims the benefit of priority to U.S. ProvisionalApplication Ser. No. 61/450,334, filed Mar. 8, 2011. The specificationsof each of the foregoing applications are incorporated herein byreference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Nov. 5, 2013, is namedALXN160102_Seq.txt, and is 50,426 bytes in size.

TECHNICAL FIELD

The field of the invention is medicine, immunology, molecular biology,and protein chemistry.

BACKGROUND

The complement system acts in conjunction with other immunologicalsystems of the body to defend against intrusion of cellular and viralpathogens. There are at least 25 complement proteins, which are found asa complex collection of plasma proteins and membrane cofactors. Theplasma proteins make up about 10% of the globulins in vertebrate serum.Complement components achieve their immune defensive functions byinteracting in a series of intricate but precise enzymatic cleavage andmembrane binding events. The resulting complement cascade leads to theproduction of products with opsonic, immunoregulatory, and lyticfunctions. A concise summary of the biologic activities associated withcomplement activation is provided, for example, in The Merck Manual,16^(th) Edition.

The complement cascade can progress via the classical pathway (CP), thelectin pathway, or the alternative pathway (AP). The lectin pathway istypically initiated with binding of mannose-binding lectin (MBL) to highmannose substrates. The AP can be antibody independent, and can beinitiated by certain molecules on pathogen surfaces. The CP is typicallyinitiated by antibody recognition of, and binding to, an antigenic siteon a target cell. These pathways converge at the C3 convertase—the pointwhere complement component C3 is cleaved by an active protease to yieldC3a and C3b.

The AP C3 convertase is initiated by the spontaneous hydrolysis ofcomplement component C3, which is abundant in the plasma in the blood.This process, also known as “tickover,” occurs through the spontaneouscleavage of a thioester bond in C3 to form C3i or C3(H₂O). Tickover isfacilitated by the presence of surfaces that support the binding ofactivated C3 and/or have neutral or positive charge characteristics(e.g., bacterial cell surfaces). This formation of C3(H₂O) allows forthe binding of plasma protein Factor B, which in turn allows Factor D tocleave Factor B into Ba and Bb. The Bb fragment remains bound to C3 toform a complex containing C3(H₂O)Bb—the “fluid-phase” or “initiation” C3convertase. Although only produced in small amounts, the fluid-phase C3convertase can cleave multiple C3 proteins into C3a and C3b and resultsin the generation of C3b and its subsequent covalent binding to asurface (e.g., a bacterial surface). Factor B bound to the surface-boundC3b is cleaved by Factor D to thus form the surface-bound AP C3convertase complex containing C3b,Bb. (See, e.g., Müller-Eberhard (1988)Ann Rev Biochem 57:321-347.)

The AP C5 convertase—(C3b)₂,Bb—is formed upon addition of a second C3bmonomer to the AP C3 convertase. (See, e.g., Medicus et al. (1976) J ExpMed 144:1076-1093 and Fearon et al. (1975) J Exp Med 142:856-863.) Therole of the second C3b molecule is to bind C5 and present it forcleavage by Bb. (See, e.g., Isenman et al. (1980) J Immunol124:326-331.) The AP C3 and C5 convertases are stabilized by theaddition of the trimeric protein properdin as described in, e.g.,Medicus et al. (1976), supra. However, properdin binding is not requiredto form a functioning alternative pathway C3 or C5 convertase. See,e.g., Schreiber et al. (1978) Proc Natl Acad Sci USA 75: 3948-3952 andSissons et al. (1980) Proc Natl Acad Sci USA 77: 559-562.

The CP C3 convertase is formed upon interaction of complement componentC1, which is a complex of C1q, C1r, and C1s, with an antibody that isbound to a target antigen (e.g., a microbial antigen). The binding ofthe C1q portion of C1 to the antibody-antigen complex causes aconformational change in C1 that activates C1r. Active C1r then cleavesthe C1-associated C1s to thereby generate an active serine protease.Active C1s cleaves complement component C4 into C4b and C4a. Like C3b,the newly generated C4b fragment contains a highly reactive thiol thatreadily forms amide or ester bonds with suitable molecules on a targetsurface (e.g., a microbial cell surface). C1s also cleaves complementcomponent C2 into C2b and C2a. The complex formed by C4b and C2a is theCP C3 convertase, which is capable of processing C3 into C3a and C3b.The CP C5 convertase—C4b, C2a, C3b—is formed upon addition of a C3bmonomer to the CP C3 convertase. See, e.g., Müller-Eberhard (1988),supra and Cooper et al. (1970) J Exp Med 132:775-793.

In addition to its role in C3 and C5 convertases, C3b also functions asan opsonin through its interaction with complement receptors present onthe surfaces of antigen-presenting cells such as macrophages anddendritic cells. The opsonic function of C3b is generally considered tobe one of the most important anti-infective functions of the complementsystem. Patients with genetic lesions that block C3b function are proneto infection by a broad variety of pathogenic organisms, while patientswith lesions later in the complement cascade sequence, i.e., patientswith lesions that block C5 functions, are found to be more prone only toNeisseria infection, and then only somewhat more prone.

The AP and CP C5 convertases cleave C5, which is a 190 kDa beta globulinfound in normal human serum at approximately 75 μg/ml (0.4 μM). C5 isglycosylated, with about 1.5-3 percent of its mass attributed tocarbohydrate. Mature C5 is a heterodimer of a 999 amino acid 115 kDaalpha chain that is disulfide linked to a 655 amino acid 75 kDa betachain. C5 is synthesized as a single chain precursor protein product ofa single copy gene (Haviland et al. (1991) J Immunol 146:362-368). ThecDNA sequence of the transcript of this gene predicts a secreted pro-C5precursor of 1658 amino acids along with an 18 amino acid leadersequence (see, e.g., U.S. Pat. No. 6,355,245).

The pro-C5 precursor is cleaved after amino acids 655 and 659, to yieldthe beta chain as an amino terminal fragment (amino acid residues +1 to655 of the above sequence) and the alpha chain as a carboxyl terminalfragment (amino acid residues 660 to 1658 of the above sequence), withfour amino acids (amino acid residues 656-659 of the above sequence)deleted between the two.

C5a is cleaved from the alpha chain of C5 by either alternative orclassical C5 convertase as an amino terminal fragment comprising thefirst 74 amino acids of the alpha chain (i.e., amino acid residues660-733 of the above sequence). Approximately 20 percent of the 11 kDamass of C5a is attributed to carbohydrate. The cleavage site forconvertase action is at, or immediately adjacent to, amino acid residue733 of the above sequence. A compound that would bind at, or adjacent,to this cleavage site would have the potential to block access of the C5convertase enzymes to the cleavage site and thereby act as a complementinhibitor. A compound that binds to C5 at a site distal to the cleavagesite could also have the potential to block C5 cleavage, for example, byway of steric hindrance-mediated inhibition of the interaction betweenC5 and the C5 convertase. A compound, in a mechanism of actionconsistent with that of the tick saliva complement inhibitor OmCI, mayalso prevent C5 cleavage by reducing flexibility of the C345C domain ofthe alpha chain of C5, which reduces access of the C5 convertase to thecleavage site of C5. See, e.g., Fredslund et al. (2008) Nat Immunol9(7):753-760.

C5 can also be activated by means other than C5 convertase activity.Limited trypsin digestion (see, e.g., Minta and Man (1997) J Immunol119:1597-1602 and Wetsel and Kolb (1982) J Immunol 128:2209-2216) andacid treatment (Yamamoto and Gewurz (1978) J Immunol 120:2008 andDamerau et al. (1989) Molec Immunol 26:1133-1142) can also cleave C5 andproduce active C5b.

Cleavage of C5 releases C5a, a potent anaphylatoxin and chemotacticfactor, and leads to the formation of the lytic terminal complementcomplex, C5b-9. C5a and C5b-9 also have pleiotropic cell activatingproperties, by amplifying the release of downstream inflammatoryfactors, such as hydrolytic enzymes, reactive oxygen species,arachidonic acid metabolites and various cytokines.

The first step in the formation of the terminal complement complexinvolves the combination of C5b with C6, C7, and C8 to form the C5b-8complex at the surface of the target cell. Upon the binding of the C5b-8complex with several C9 molecules, the membrane attack complex (MAC,C5b-9, terminal complement complex—TCC) is formed. When sufficientnumbers of MACs insert into target cell membranes the openings theycreate (MAC pores) mediate rapid osmotic lysis of the target cells.Lower, non-lytic concentrations of MACs can produce other effects. Inparticular, membrane insertion of small numbers of the C5b-9 complexesinto endothelial cells and platelets can cause deleterious cellactivation. In some cases activation may precede cell lysis.

As mentioned above, C3a and C5a are anaphylatoxins. These activatedcomplement components can trigger mast cell degranulation, whichreleases histamine from basophils and mast cells, and other mediators ofinflammation, resulting in smooth muscle contraction, increased vascularpermeability, leukocyte activation, and other inflammatory phenomenaincluding cellular proliferation resulting in hypercellularity. C5a alsofunctions as a chemotactic peptide that serves to attractpro-inflammatory granulocytes to the site of complement activation.

C5a receptors are found on the surfaces of bronchial and alveolarepithelial cells and bronchial smooth muscle cells. C5a receptors havealso been found on eosinophils, mast cells, monocytes, neutrophils, andactivated lymphocytes.

While a properly functioning complement system provides a robust defenseagainst infecting microbes, inappropriate regulation or activation ofcomplement has been implicated in the pathogenesis of a variety ofdisorders including, e.g., rheumatoid arthritis (RA); lupus nephritis;asthma; ischemia-reperfusion injury; atypical hemolytic uremic syndrome(aHUS); dense deposit disease (DDD); paroxysmal nocturnal hemoglobinuria(PNH); macular degeneration (e.g., age-related macular degeneration(AMD)); hemolysis, elevated liver enzymes, and low platelets (HELLP)syndrome; thrombotic thrombocytopenic purpura (TTP); spontaneous fetalloss; Pauci-immune vasculitis; epidermolysis bullosa; recurrent fetalloss; multiple sclerosis (MS); traumatic brain injury; and injuryresulting from myocardial infarction, cardiopulmonary bypass andhemodialysis. (See, e.g., Holers et al. (2008) Immunological Reviews223:300-316.) Inhibition of complement (e.g., inhibition of: terminalcomplement formation, C5 cleavage, or complement activation) has beendemonstrated to be effective in treating several complement-associateddisorders both in animal models and in humans. See, e.g., Rother et al.(2007) Nature Biotechnology 25(11):1256-1264; Wang et al. (1996) ProcNatl Acad Sci USA 93:8563-8568; Wang et al. (1995) Proc Natl Acad SciUSA 92:8955-8959; Rinder et al. (1995) J Clin Invest 96:1564-1572;Kroshus et al. (1995) Transplantation 60:1194-1202; Homeister et al.(1993) J Immunol 150:1055-1064; Weisman et al. (1990) Science249:146-151; Amsterdam et al. (1995) Am J Physiol 268:H448-H457; andRabinovici et al. (1992) J Immunol 149:1744 1750.

SUMMARY

This disclosure relates to stable, highly-concentrated liquidformulations of antibodies as well as methods for making and using theformulations. The disclosure provides, among other things, formulationconditions suitable for maintaining over considerable time the physicaland functional stability of an anti-C5 antibody (e.g., eculizumab) inhigh concentration solutions. For example, the disclosure providesformulation conditions capable of maintaining an anti-C5 antibody inpredominantly monomeric form for up to 2 years at 2° C. to 8° C., evenwhen the antibody is maintained in solutions at concentrations ofapproximately 100 mg/mL. In addition, as described herein andexemplified in the working examples, such formulations also minimizeaggregation, fragmentation, or degradation of an anti-C5 antibody withinthe highly-concentrated solutions. For example, the disclosure providesformulation conditions capable of maintaining for two years an anti-C5antibody in a highly-concentrated form with no detectable antibodyfragmentation or degradation products (as determined using sizeexclusion chromatography-high performance liquid chromatography(SEC-HPLC)) and no more than 2% aggregate. Also provided herein areconditions suitable for formulating solutions of an anti-C5 antibodysuch as eculizumab at greater than 200 mg/mL.

The benefits of stable, highly-concentrated aqueous solutions of ananti-C5 antibody are numerous. First, for therapeutic applications whichrequire the antibody to be administered to a patient in a small volume,therapeutic efficacy often turns on the amount of antibody that can beadministered in that small volume. In the absence of the ability toformulate an anti-C5 antibody to high concentrations, use of, forexample, subcutaneous, intravitreal, and/or intraarticular deliveryroutes would often be precluded. Relatedly, highly-concentrated antibodyformulations allow for more patient choice regarding the route ofadministration. For therapeutic applications that require frequentand/or chronic administration, self-delivery or -administration is madepossible by high concentration formulations and can be more appealing topatients than intravenous infusion. For example, high concentrationformulations of an anti-C5 antibody can allow a patient toself-administer the antibody by, e.g., subcutaneous injection.Therefore, the ability to formulate the antibody at high concentrationscan increase compliance of administration by providing an easy homeadministration alternative to patients with complement-associateddisorders.

Furthermore, methods for producing the aqueous solutions describedherein do not require a lyophilization step, nor do the featured highconcentration aqueous solutions need to be reconstituted fromlyophilized material. The instantly featured high concentration antibodysolutions provide several advantages over reconstituted lyophilizedantibody formulations. First, medical practitioners must locallyreconstitute lyophilized antibody solutions aseptically, which increasesthe opportunity for microbial contamination of the solution prior toadministration. In addition, reconstitution requires considerable careto be certain that all of the solids contained in the reconstitutionvessel are properly dissolved in solution. The high concentrationaqueous solutions provided herein thus provide the medical practitioner,caregiver, and/or patient with a fast, easy, safe, and efficient meansfor delivering a therapeutic antibody to a patient in need thereof.

Other benefits of high concentration formulations include, e.g.,manufacturing cost savings from decreasing bulk storage space and/or thenumber of product fills. In addition, the ability to produce a producthaving a longer shelf-life will ultimately require fewer productionruns, which ultimately reduces cost for the manufacturer and consumer ofthe highly-concentrated therapeutic antibody.

In one aspect, the disclosure features an aqueous solution comprising ananti-C5 antibody at a concentration of 40 mg/mL to 200 mg/mL. In anotheraspect, the disclosure features an aqueous solution comprising ananti-C5 antibody at a concentration of greater than 200 mg/mL.Additional exemplary concentrations, including fixed concentrations aswell as exemplary ranges of concentrations, are provided herein.

In some embodiments, any of the solutions described herein comprise atleast one buffering agent at a concentration of 10 mM to 300 mM,inclusive. In some embodiments, any of the solutions described hereincomprise at least one buffering agent at a concentration of 10 mM to 200mM, inclusive. In some embodiments, the at least one buffering agent ispresent in the solution at a concentration of at least, or equal to, 20mM. In some embodiments, the at least one buffering agent is present inthe solution at a concentration of at least, or equal to, 50 mM. In someembodiments, the at least one buffering agent is an amino acid. Theamino acid can be, e.g., one selected from the group consisting ofhistidine (e.g., L-histidine), serine (e.g., L-serine), and glycine(e.g., L-glycine). In some embodiments, any of the solutions describedherein comprise two or more buffering agents. The two or more bufferingagents can be, e.g., histidine and serine. In some embodiments, the twoor more buffering agents are histidine and glycine.

In some embodiments, any of the solutions described herein comprise atleast one carbohydrate excipient at a concentration of 0.1 to 5%. Insome embodiments, the at least one carbohydrate excipient is present inthe solution at a concentration of at least, or equal to, 1.5%. In someembodiments, the at least one carbohydrate excipient is present in thesolution at a concentration of at least, or equal to, 3%. The at leastone carbohydrate excipient can be, e.g., one selected from the groupconsisting of sorbitol and mannitol. In some embodiments, any of thesolutions described herein comprise two or more carbohydrate excipients.At least two of the excipients can be, e.g., sorbitol and mannitol.

In some embodiments, any of the solutions described herein comprise aformulation that comprises, or consists of, the following composition:(i) at least 20 mM histidine; at least 50 mM glycine; at least 3% (w/v)sorbitol; and at least 1.5% (w/v) mannitol; (ii) 20 mM histidine; 50 mMglycine; 3% (w/v) sorbitol; and 1.5% (w/v) mannitol; (iii) at least 20mM histidine; at least 50 mM serine; at least 3% (w/v) sorbitol; and atleast 1.5% (w/v) mannitol; or (iv) at least 20 mM histidine; at least 50mM serine; at least 2.5% (w/v) sorbitol; and at least 1.5% (w/v)mannitol. Additional exemplary formulations are set forth herein.

In some embodiments, any of the solutions described herein comprise asurfactant. The surfactant can be, e.g., polysorbate 20 or polysorbate80. The concentration of the surfactant in the solution can be, e.g.,between 0.001% to 0.02%, inclusive.

In some embodiments, any of the solutions described herein can have a pHbetween 6.5 and 7.5.

In some embodiments, any of the solutions described herein are sterilesolutions.

In some embodiments of any of the solutions described herein, theanti-C5 antibody is eculizumab.

In some embodiments of any of the solutions described herein, theanti-C5 antibody remains at least 95 (e.g., at least 96, 97, 98, or 99)% monomeric during storage at 2° C. to 8° C. for at least six months asdetermined by SEC-HPLC. In some embodiments of any of the solutionsdescribed herein, the anti-C5 antibody remains at least 95 (e.g., atleast 96, 97, 98, or 99) % monomeric during storage at 2° C. to 8° C.for at least one year as determined by SEC-HPLC. In some embodiments ofany of the solutions described herein, the anti-C5 antibody remains atleast 95 (e.g., at least 96, 97, 98, or 99) % monomeric during storageat 2° C. to 8° C. for at least six months as determined by SEC-HPLC. Insome embodiments of any of the solutions described herein, the anti-C5antibody remains at least 95 (e.g., at least 96, 97, 98, or 99) %monomeric during storage at 2° C. to 8° C. for at least one year asdetermined by SEC-HPLC. In some embodiments of any of the solutionsdescribed herein, the anti-C5 antibody remains at least 95 (e.g., atleast 96, 97, 98, or 99) % monomeric during storage at 2° C. to 8° C.for at least 18 months as determined by SEC-HPLC. In some embodiments ofany of the solutions described herein, the anti-C5 antibody remains atleast 95 (e.g., at least 96, 97, 98, or 99) % monomeric during storageat 2° C. to 8° C. for at least two years as determined by SEC-HPLC.

In some embodiments of any of the solutions described herein, less than2% of the anti-C5 antibody in the solution is aggregated as determinedby SEC-HPLC. In some embodiments of any of the solutions describedherein, less than 1% of the anti-C5 antibody in the solution isaggregated as determined by SEC-HPLC.

In some embodiments of any of the solutions described herein, less than1% of the anti-C5 antibody in the solution is fragmented as determinedby SEC-HPLC. In some embodiments of any of the solutions describedherein, less than 0.5% of the anti-C5 antibody in the solution isfragmented as determined by SEC-HPLC. In some embodiments of any of thesolutions described herein, during storage at 2° C. to 8° C. for atleast six months the anti-C5 antibody retains at least 80 (e.g., atleast 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, or 99) % of its C5-binding activity, as compared to a referenceanti-C5 antibody corresponding to the anti-C5 antibody prior to storage.In some embodiments of any of the solutions described herein, duringstorage at 2° C. to 8° C. for at least one year the anti-C5 antibodyretains at least 80 (e.g., at least 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, or 99) % of its C5-binding activity,as compared to a reference anti-C5 antibody corresponding to the anti-C5antibody prior to storage. In some embodiments of any of the solutionsdescribed herein, during storage at 2° C. to 8° C. for at least 18months the anti-C5 antibody retains at least 80 (e.g., at least 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99) %of its C5-binding activity, as compared to a reference anti-C5 antibodycorresponding to the anti-C5 antibody prior to storage. In someembodiments of any of the solutions described herein, during storage at2° C. to 8° C. for at least two years the anti-C5 antibody retains atleast 80 (e.g., at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, or 99) % of its C5-binding activity, as comparedto a reference anti-C5 antibody corresponding to the anti-C5 antibodyprior to storage. In some embodiments of any of the solutions describedherein, during storage at 2° C. to 8° C. for at least six months theanti-C5 antibody retains at least 80 (e.g., at least 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99) % of itsability to inhibit hemolysis, as compared to a reference anti-C5antibody corresponding to the anti-C5 antibody prior to storage. In someembodiments of any of the solutions described herein, during storage at2° C. to 8° C. for at least one year the anti-C5 antibody retains atleast 80 (e.g., at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, or 99) % of its ability to inhibit hemolysis, ascompared to a reference anti-C5 antibody corresponding to the anti-C5antibody prior to storage. In some embodiments of any of the solutionsdescribed herein, during storage at 2° C. to 8° C. for at least 18months the anti-C5 antibody retains at least 80 (e.g., at least 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99) %of its ability to inhibit hemolysis, as compared to a reference anti-C5antibody corresponding to the anti-C5 antibody prior to storage. In someembodiments of any of the solutions described herein, during storage at2° C. to 8° C. for at least two years the anti-C5 antibody retains atleast 80 (e.g., at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, or 99) % of its ability to inhibit hemolysis, ascompared to a reference anti-C5 antibody corresponding to the anti-C5antibody prior to storage.

In another aspect, the disclosure features an aqueous solutioncomprising an anti-C5 antibody at a concentration of 100±20 (e.g., 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, or 120) mg/mL; 20±5 (e.g., 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25) mM L-histidine; 50±15 (e.g., 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65) mM L-serine; 3±1(e.g., 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3,3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4) % sorbitol; and 1.5±0.5 (e.g., 1,1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2) % mannitol, whereinthe solution has a pH of 7.1±0.5 (e.g., 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2,7.3, 7.4, 7.5, or 7.6).

In yet another aspect, the disclosure features a method for producing aconcentrated antibody solution comprising greater than (or equal to) 100mg/mL of an anti-C5 antibody. The method comprises: providing a firstaqueous solution comprising an anti-C5 antibody, the first aqueoussolution having a first formulation and comprising no more than 50(e.g., no more than 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37,36, 35, 34, 33, 32, or 31) mg/mL of the anti-C5 antibody; subjecting thefirst aqueous solution to diafiltration to thereby produce a secondaqueous solution, wherein the second aqueous solution has a secondformulation as a result of the diafiltration; and concentrating thesecond aqueous solution to produce a concentrated antibody solutioncomprising greater than (or equal to) 100 mg/mL of the anti-C5 antibody.In some embodiments, the first aqueous solution comprises greater than30 mg/mL, but no more than 50 mg/mL, of the anti-C5 antibody. In someembodiments, the first aqueous solution comprises greater than 35 mg/mL,but no more than 50 mg/mL, of the anti-C5 antibody. In some embodiments,the first aqueous solution comprises greater than 35 mg/mL, but no morethan 45 mg/mL, of the anti-C5 antibody. In some embodiments, the anti-C5antibody is not lyophilized prior to or following the diafiltration orconcentrating.

In some embodiments of any of the methods, the first formulation is aphosphate buffer-based formulation. The first formulation can comprise,e.g.: at least 20 mM sodium phosphate and at least 80 mM sodiumchloride.

In some embodiments of any of the above methods, the second formulationcomprises: at least 20 mM histidine; at least 50 mM serine; at least2.5% (w/v) sorbitol; and at least 1.5% (w/v) mannitol.

In some embodiments of any of the above methods, the concentratingcomprises tangential flow filtration and/or use of a stir cell.

In some embodiments of any of the above methods, more than one round ofdiafiltration is performed. In some embodiments, at least two rounds ofdiafiltration are performed. In some embodiments, at least four roundsof diafiltration are performed. In some embodiments of any of the abovemethods, the diafiltration comprises continuous addition of a bufferhaving the second formulation.

In some embodiments of any of the above methods, the concentratedantibody solution comprises greater than (or equal to) 105 (e.g.,greater than, or equal to, 106, 107, 108, 109, 110, 115, 120, 125, 130,135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200,205, or 208) mg/mL of the anti-C5 antibody.

In some embodiments of any of the above methods, at least 80 (e.g., atleast 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, or 99) % of the anti-C5 antibody present in the first aqueoussolution is recovered in the high concentration aqueous solution. Insome embodiments of any of the above methods, at least 90% of theanti-C5 antibody present in the first aqueous solution is recovered inthe high concentration aqueous solution.

In some embodiments of any of the above methods, the anti-C5 antibody iseculizumab.

In yet another aspect, the disclosure features an aqueous solutioncomprising an anti-C5 antibody at a concentration of greater than 100mg/mL produced by any of the above methods.

In another aspect, the disclosure features a kit comprising: (i) any ofthe solutions described herein; and (ii) a means for delivering thesolution to a patient in need thereof.

In some embodiments of any of the kits described herein, the means issuitable for subcutaneous delivery of the solution to the patient. Insome embodiments of any of the kits described herein, the means issuitable for delivery of the solution to the eye. In some embodiments ofany of the kits described herein, the means is suitable forintraarticular delivery of the solution to the patient.

In some embodiments of any of the kits described herein, the means is asyringe or a double-barreled syringe. In some embodiments of any of thekits described herein, the means is: (a) a transscleral patch comprisingthe solution; or (b) a contact lens comprising the solution or partiallycoated in the solution.

In some embodiments of any of the kits described herein, the means issuitable for intrapulmonary delivery of the solution to the patient. Forexample, the means can be an inhaler or a nebulizer.

In some embodiments of any of the kits described herein, the solution isformulated for aerosol administration or nebulized administration to thepatient.

In some embodiments, any of the kits described herein further compriseat least one additional active agent for use in treating acomplement-associated disorder in a subject. Such agents are recitedherein.

In yet another aspect, the disclosure features a kit comprising one ormore containers, wherein each container comprises an aqueous solutiondescribed herein and wherein each container comprises at least onepharmaceutical unit dosage form of the anti-C5 antibody. In someembodiments, each container comprises between 0.05 mg to 10 mg of theanti-C5 antibody. In some embodiments, the kit comprises between about 1mg and 100 mg of the anti-C5 antibody.

In some embodiments of any of the kits described herein, each containerhas a volume of 0.01 mL to 1 mL, inclusive. In some embodiments of anyof the kits described herein, at least one container comprises anaqueous solution suitable for intravitreal injection to a patient,

intraarticular injection to a patient, intramuscular injection to apatient, subcutaneous injection to a patient, and/or intrapulmonaryadministration to a patient. For example, a kit described herein cancomprise at least one container comprising an aqueous solution suitablefor use with a nebulizer or inhaler.

In another aspect, the disclosure features a pre-filled syringecomprising any of the aqueous solutions described herein. In someembodiments, the solution is formulated for intraocular, intravitreal,and/or intraarticular administration. In some embodiments, the solutionis formulated for intramuscular or subcutaneous administration.

In some embodiments, any of the pre-filled syringes described hereincomprise at least one pharmaceutical unit dosage form of the anti-C5antibody in the solution. Each pharmaceutical unit dosage form can have,e.g., a volume of between 0.02 mL to 0.1 mL, inclusive. In someembodiments, the pharmaceutical unit dosage form has a volume of no morethan 0.05 mL.

In some embodiments, any of the pre-filled syringes described hereincomprise between 0.05 mg to 10 mg of the anti-C5 antibody. In someembodiments, the syringe comprises between about 1 mg and 100 mg of theanti-C5 antibody.

In yet another aspect, the disclosure features a method for treating apatient afflicted with a complement-associated disorder. The methodcomprises administering to a patient afflicted with acomplement-associated disorder a therapeutically effective amount of anyof the aqueous solutions described herein to thereby treat thecomplement-associated disorder. In some embodiments, the methods can beperformed using any of the kits or pre-filled syringes described herein.In some embodiments, the method can further comprise, prior toadministering the aqueous solution to the patient, determining that thepatient is afflicted with the complement-associated disorder.

In some embodiments, the complement-associated disorder is acomplement-associated disorder of the eye. For example, thecomplement-associated disorder of the eye can be age-related maculardegeneration (AMD), a diabetes-associated ocular disorder, or centralretinal vein occlusion. In some embodiments, the complement-associateddisorder of the eye is wet AMD. In some embodiments, the disorder is dryAMD. In such embodiments, the aqueous solution can be administered tothe patient by way of intravitreal injection. In such embodiments, theaqueous solution can be administered to the patient by way of atransscleral patch or as an eye drop (for example, the solution can beformulated for use as an eye drop).

In some embodiments, the complement-associated disorder is rheumatoidarthritis. In such embodiments, e.g., the aqueous solution can beadministered to the patient by way of intraarticular injection. In someembodiments, the aqueous solution can be administered by way ofintravenous or subcutaneous injection.

In some embodiments, the complement-associated disorder is a pulmonarydisorder. The pulmonary disorder can be selected from the groupconsisting of, e.g., asthma, chronic obstructive pulmonary disease,acute respiratory distress syndrome, pulmonary fibrosis, α-1anti-trypsin deficiency, emphysema, bronchiectasis, bronchiolitisobliterans, sarcoidosis, a collagen vascular disorder, and bronchitis.In such embodiments, the aqueous solution can be delivered to thepatient by way of intrapulmonary administration, e.g., through the useof a nebulizer or an inhaler.

In some embodiments, the complement-associated disorder is selected fromthe group consisting of ischemia-reperfusion injury, atypical hemolyticuremic syndrome, thrombotic thrombocytopenic purpura, paroxysmalnocturnal hemoglobinuria, dense deposit disease, age-related maculardegeneration, spontaneous fetal loss, Pauci-immune vasculitis,epidermolysis bullosa, recurrent fetal loss, multiple sclerosis,traumatic brain injury, myasthenia gravis, cold agglutinin disease,dermatomyositis, Degos' disease, Graves' disease, Hashimoto'sthyroiditis, type I diabetes, psoriasis, pemphigus, autoimmune hemolyticanemia, idiopathic thrombocytopenic purpura, Goodpasture syndrome,multifocal motor neuropathy, neuromyelitis optica, antiphospholipidsyndrome, and catastrophic antiphospholipid syndrome.

In some embodiments, any of the therapeutic methods described hereinfurther comprise administering to the patient one or more additionaltherapeutic agents for (a) treating a complement-associated disorder or(b) ameliorating one or more symptoms associated with thecomplement-associated disorder.

In yet another embodiment, the disclosure features an aqueous solutioncomprising an anti-C5 antibody at a concentration of at least 40 mg/mL.The anti-C5 antibody in the solution remains at least 97% monomericduring storage at 2° C. to 8° C. for at least six months as determinedby SEC-HPLC. In some embodiments, the concentration of the anti-C5antibody in the solution is at least 50 (e.g., at least 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or200) mg/mL. In some embodiments, the concentration of the anti-C5antibody in the solution is greater than 200 mg/mL. The antibody can be,e.g., eculizumab.

In another aspect, the disclosure features an aqueous solutioncomprising an anti-C5 antibody at a concentration of greater than 40mg/mL with the proviso that the solution is not formulated as follows:20 mM histidine, 50 mM glycine, 3% (w/v) sorbitol, 1.5% (w/v) mannitol,0.001% to 0.02% Tween 80, and a pH of 6 to 8 (e.g., with a physiologicosmolality). In some embodiments, the concentration of the anti-C5antibody in the solution is at least 50 (e.g., at least 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or200) mg/mL. In some embodiments, the concentration of the anti-C5antibody in the solution is greater than 200 mg/mL. The antibody can be,e.g., eculizumab.

In some embodiments of the aqueous solutions described above, theanti-C5 antibody remains at least 98% monomeric during storage at 2° C.to 8° C. for at least one year (e.g., at least 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, or 24 months) as determined by SEC-HPLC.

In some embodiments of the aqueous solutions described above, less than2 (e.g., less than 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0,0.9, 0.8. 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2) % of the anti-C5 antibody inthe solution is aggregated as determined by SEC-HPLC.

In some embodiments of the aqueous solutions described above, less than0.5 (e.g., less than 0.4, 0.3, 0.2, or 0.1) % of the anti-C5 antibody inthe solution is fragmented as determined by SEC-HPLC.

In some embodiments of the aqueous solutions described above, duringstorage at 2° C. to 8° C. for at least six (e.g., at least seven, eight,nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24)months the anti-C5 antibody retains at least 90 (e.g., at least 91, 92,93, 94, 95, 96, 97, 98, or 99) % of its C5-binding activity, as comparedto a reference anti-C5 antibody corresponding to the anti-C5 antibodyprior to storage. Suitable methods for evaluating the binding activityof a sample of a stored solution containing a specified concentration ofanti-C5 antibody are known in the art and described herein.

In some embodiments of the aqueous solutions described above, duringstorage at 2° C. to 8° C. for at least six (e.g., at least seven, eight,nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24)months the anti-C5 antibody retains at least 90 (e.g., at least 91, 92,93, 94, 95, 96, 97, 98, or 99) % of its ability to inhibit hemolysis, ascompared to a reference anti-C5 antibody corresponding to the anti-C5antibody prior to storage. Suitable methods for evaluating the abilityof a sample of a stored solution containing a specified concentration ofanti-C5 antibody to inhibit hemolysis are known in the art and describedherein.

In some embodiments of the aqueous solutions described above, thesolutions can be sterile. In some embodiments of any of the aqueoussolutions described above, the solutions can comprise: at least 20 mMhistidine; at least 50 mM serine; at least 3% (w/v) sorbitol; and atleast 1.5% (w/v) mannitol. In some embodiments of the aqueous solutionsdescribed above, the solutions can comprise: at least 20 mM histidine;at least 50 mM serine; at least 2.5% (w/v) sorbitol; and at least 1.5%(w/v) mannitol. In some embodiments of any of the aqueous solutionsdescribed above, the solutions can contain: 20 mM histidine; 50 mMserine; 3% (w/v) sorbitol; and 1.5% (w/v) mannitol.

In some embodiments of the aqueous solutions described above, thesolution can comprise a surfactant such as, for example, polysorbate 20or polysorbate 80. The concentration of the surfactant in the solutioncan be, e.g., between 0.001% to 0.02%, inclusive.

In some embodiments of the aqueous solutions described above, the pH ofthe solution can be, for example, between 6.5 and 7.5, inclusive.

As used throughout the present disclosure, the term “antibody” refers toa whole antibody (e.g., IgM, IgG, IgA, IgD, or IgE) molecule as well asantigen-binding fragments thereof, which fragments can be generated byany one of a variety of methods that are known in the art and describedherein. The term “antibody” includes a polyclonal antibody, a monoclonalantibody, a chimerized or chimeric antibody, a humanized antibody, adeimmunized human antibody, and a fully human antibody. The antibody canbe made in or derived from any of a variety of species, e.g., mammalssuch as humans, non-human primates (e.g., monkeys, baboons, orchimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits,guinea pigs, gerbils, hamsters, rats, and mice. The antibody can be apurified or a recombinant antibody. “Antibody fragments,”“antigen-binding fragments,” or similar terms refer to a fragment of anantibody that retains the ability to bind to an antigen (e.g., humancomplement component C5 or a biologically active fragment thereof suchas C5a or C5b), e.g., a single chain antibody, a single chain Fvfragment (scFv), an Fd fragment, an Fab fragment, an Fab′ fragment, oran F(ab′)₂ fragment. An scFv fragment is a single polypeptide chain thatincludes both the heavy and light chain variable regions of the antibodyfrom which the scFv is derived. In addition, diabodies (Poljak (1994)Structure 2(12):1121-1123; Hudson et al. (1999) J Immunol Methods23(1-2):177-189, the disclosures of both of which are incorporatedherein by reference in their entirety), minibodies, single domain ornanobodies (Huang et al. (2010) Expert Rev Mol Diagn 10(6):777-785;Smolarek et al. (2010) Cell Mol Life Sci 67(19):3371-3387), andintrabodies (Huston et al. (2001) Hum Antibodies 10(3-4):127-142;Wheeler et al. (2003) Mol Ther 8(3):355-366; Stocks (2004) Drug DiscovToday 9(22):960-966, the disclosures of each of which are incorporatedherein by reference in their entirety) that bind to human C5 protein areembraced by the definition of “antigen-binding fragment” and can beincorporated into the compositions and used in the methods describedherein.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure pertains. In case of conflict, thepresent document, including definitions, will control. Preferred methodsand materials are described below, although methods and materialssimilar or equivalent to those described herein can also be used in thepractice or testing of the presently disclosed methods and compositions.All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Other features and advantages of the present disclosure, e.g., methodsfor treating a complement-associated disorder, will be apparent from thefollowing description, the examples, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart depicting the formulation scheme for preparingfive different solutions of eculizumab. A detailed description of theparticular composition of solutions I to V, as well as the nature of theHSSM and HTT buffers is set forth in Example 2 (infra).

DETAILED DESCRIPTION

The disclosure features stable, aqueous solutions containing a highconcentration of an antibody that binds to human complement componentC5. The solutions can be used in a variety of therapeutic applicationssuch as methods for treating or preventing complement-associateddisorders. While in no way intended to be limiting, exemplary solutions,formulations, therapeutic kits, and methods for making and using any ofthe foregoing are elaborated on below and are exemplified in the workingExamples.

Highly-Concentrated Antibody Solutions

The disclosure provides aqueous solutions comprising a highconcentration of an antibody that binds to human complement component C5[hereinafter an “anti-C5 antibody” or an “anti-human C5 antibody”] suchas eculizumab. Such solutions are sometimes referred to herein as “highconcentration antibody solutions.” As used herein, a “highconcentration” of an anti-C5 antibody in an aqueous solution is aconcentration of the antibody that is at least, equal to, or greaterthan, 40 (e.g., at least, equal to, or greater than, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140,145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210,215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280,285, or 290) mg/mL. In some embodiments, the anti-C5 antibody is presentin the solution at a concentration of more than 200 (e.g., more than200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265,270, 275, 280, 285, or 290) mg/mL. In some embodiments, the antibody ispresent in the solution at a concentration of, e.g., 40 mg/mL to 200mg/mL, 50 mg/mL to 200 mg/mL, 60 mg/mL to 200 mg/mL, 70 mg/mL to 200mg/mL, 80 mg/mL to 200 mg/mL, 90 mg/mL to 200 mg/mL, 100 mg/mL to 200mg/mL, 110 mg/mL to 200 mg/mL, 120 mg/mL to 200 mg/mL, 130 mg/mL to 200mg/mL, 140 mg/mL to 200 mg/mL, 150 mg/mL to 200 mg/mL, 40 mg/mL to 100mg/mL, 50 mg/mL to 100 mg/mL, 60 mg/mL to 100 mg/mL, 70 mg/mL to 100mg/mL, 80 mg/mL to 100 mg/mL, 90 mg/mL to 100 mg/mL, 40 mg/mL to 150mg/mL, 50 mg/mL to 150 mg/mL, 60 mg/mL to 150 mg/mL, 70 mg/mL to 150mg/mL, 80 mg/mL to 150 mg/mL, 90 mg/mL to 150 mg/mL, 100 mg/mL to 150mg/mL, 110 mg/mL to 150 mg/mL, 120 mg/mL to 150 mg/mL, 40 mg/mL to 50mg/mL, 40 mg/mL to 250 mg/mL, 50 mg/mL to 250 mg/mL, 60 mg/mL to 250mg/mL, 70 mg/mL to 250 mg/mL, 80 mg/mL to 250 mg/mL, 90 mg/mL to 250mg/mL, 100 mg/mL to 250 mg/mL, 110 mg/mL to 250 mg/mL, 120 mg/mL to 250mg/mL, 130 mg/mL to 250 mg/mL, 140 mg/mL to 250 mg/mL, 150 mg/mL to 250mg/mL, 160 mg/mL to 250 mg/mL, 170 mg/mL to 250 mg/mL, 180 mg/mL to 250mg/mL, 190 mg/mL to 250 mg/mL, 200 mg/mL to 250 mg/mL, greater than 200mg/mL (e.g., at least 201 mg/mL) to 250 mg/mL, or greater than 200 mg/mL(e.g., 201 mg/mL or greater) to 300 mg/mL.

In some embodiments, the anti-C5 antibody binds to an epitope in thehuman pro-C5 precursor protein. For example, the anti-C5 antibody canbind to an epitope in the human complement component C5 proteincomprising, or consisting of, the amino acid sequence depicted in SEQ IDNO:1 (NCBI Accession No. AAA51925 and Haviland et al., supra).

An “epitope” refers to the site on a protein (e.g., a human complementcomponent C5 protein) that is bound by an antibody. “Overlappingepitopes” include at least one (e.g., two, three, four, five, or six)common amino acid residue(s).

In some embodiments, the anti-C5 antibody binds to an epitope in thehuman pro-C5 precursor protein lacking the leader sequence. For example,the anti-C5 antibody can bind to an epitope in the human complementcomponent C5 protein comprising, or consisting of, the amino acidsequence depicted in SEQ ID NO:2, which is a human C5 protein lackingthe amino terminal leader sequence.

In some embodiments, the anti-C5 antibody can bind to an epitope in thealpha chain of the human complement component C5 protein. For example,the anti-C5 antibody can bind to an epitope within, or overlapping with,a protein having the amino acid sequence depicted in SEQ ID NO:3, whichis the human complement component C5 alpha chain protein. Antibodiesthat bind to the alpha chain of C5 are described in, for example, Ameset al. (1994) J Immunol 152:4572-4581.

In some embodiments, the anti-C5 antibody can bind to an epitope in thebeta chain of the human complement component C5 protein. For example,the anti-C5 antibody can bind to an epitope within, or overlapping with,a protein having the amino acid sequence depicted in SEQ ID NO:4, whichis the human complement component C5 beta chain protein. Antibodies thatbind to the C5 beta chain are described in, e.g., Moongkarndi et al.(1982) Immunobiol 162:397; Moongkarndi et al. (1983) Immunobiol 165:323;and Mollnes et al. (1988) Scand J Immunol 28:307-312.

In some embodiments, the anti-C5 antibody can bind to an epitope within,or overlapping with, an antigenic peptide fragment of a human complementcomponent C5 protein. For example, the anti-C5 antibody can bind to anepitope within, or overlapping with, an antigen peptide fragment of ahuman complement component C5 protein, the fragment containing, orconsisting of, the following amino acid sequence:

(SEQ ID NO: 5) VIDHQGTKSSKCVRQKVEGSS or (SEQ ID NO: 6) KSSKC.

In some embodiments, the anti-C5 antibody can bind to an epitope within,or overlapping with, a fragment of a human complement component C5protein, the fragment containing, or consisting of, any one of thefollowing amino acid sequences (which are exemplary antigenic fragmentsof SEQ ID NO:1):

(SEQ ID NO: 7) NFSLETWFGKEILVKTLRVVPEGVKRESYSGVTLDPRGIYGTISRRKEFPYRIPLDLVPKTEIKRILSVKGLLVGEILSAVLSQEGINILTHLPKGSAEAELMSVVPVFYVFHYLETGNHWNIFHSD; (SEQ ID NO: 8)SESPVIDHQGTKSSKCVRQKVEGSSSHLVTFTVLPLEIGLHNINFSLETWFGKEILVKTLRVVPEGVKRESYSGVTLDPRGIYGTISRRKEFPYRIPLDLVPKTEIKRILSVKGLLVGEILSAVLSQEGINILTHLPKGSAEAELMSVVPVFYVFHYLETGNHWNIFHSDPLIEKQKLKKKLKEGMLSIMSYRNAD YSYS; (SEQ ID NO: 9)SHKDMQLGRLHMKTLLPVSKPEIRSYFPES; (SEQ ID NO: 10)SHKDMQLGRLHMKTLLPVSKPEIRSYFPESWLWEVHLVPRRKQLQFALPDSLTTWEIQGIGISNTGICVADTVKAKVFKDVFLEMNIPYSVVRGEQIQLKGTVYNYRTSGMQFCVKMSAVEGICTSESPVIDHQGTKSSKCVRQKVEGSSSHLVTFTVLPLEIGLHNINFSLETWFGKEILVKTLRVVPEGVKRESYSGVTLDPRGIYGTISRRKEFPYRIPLDLVPKTEIKRILSVKGLLVGEILSAVLSQEGINILTHLPKGSAEAELMSVVPVFYVFHYLETGNHWNIFHSDPLIEKQKLKKKLKEGMLSIMSYRNADYSYS; and (SEQ ID NO: 11) DHQGTKSSKCVRQKVEG.

Additional exemplary antigenic fragments of human complement componentC5 are disclosed in, e.g., U.S. Pat. No. 6,355,245, the disclosure ofwhich is incorporated herein by reference.

In some embodiments, the anti-C5 antibody specifically binds to a humancomplement component C5 protein (e.g., the human C5 protein having theamino acid sequence depicted in SEQ ID NO:1). The terms “specificbinding” or “specifically binds” refer to two molecules forming acomplex (e.g., a complex between an antibody and a complement componentC5 protein) that is relatively stable under physiologic conditions.Typically, binding is considered specific when the association constant(K_(a)) is higher than 10⁶ M⁻¹. Thus, an antibody can specifically bindto a C5 protein with a K_(a). of at least (or greater than) 10⁶ (e.g.,at least or greater than 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹ 10¹², 10¹³, 10¹⁴, or10¹⁵ or higher) M⁻¹. Examples of antibodies that specifically bind to ahuman complement component C5 protein are described in, e.g., U.S. Pat.No. 6,355,245, the disclosure of which is incorporated herein byreference in its entirety.

Methods for determining whether an antibody binds to a protein antigenand/or the affinity for an antibody to a protein antigen are known inthe art. For example, the binding of an antibody to a protein antigencan be detected and/or quantified using a variety of techniques such as,but not limited to, Western blot, dot blot, surface plasmon resonance(SPR) method (e.g., BIAcore™ system; Pharmacia Biosensor AB, Uppsala,Sweden and Piscataway, N.J.), or enzyme-linked immunosorbent assays(ELISA). See, e.g., Harlow and Lane (1988) “Antibodies: A LaboratoryManual” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;Benny K. C. Lo (2004) “Antibody Engineering: Methods and Protocols,”Humana Press (ISBN: 1588290921); Borrebaek (1992) “Antibody Engineering,A Practical Guide,” W.H. Freeman and Co., NY; Borrebaek (1995) “AntibodyEngineering,” 2^(nd) Edition, Oxford University Press, NY, Oxford; Johneet al. (1993) J Immunol Meth 160:191-198; Jonsson et al. (1993) Ann BiolClin 51:19-26; and Jonsson et al. (1991) Biotechniques 11:620-627. Seealso, U.S. Pat. No. 6,355,245.

In some embodiments, the anti-C5 antibody can crossblock binding ofanother antibody that binds to an epitope within, or overlapping with, ahuman complement component C5 protein. In some embodiments, the anti-C5antibody can crossblock binding of an antibody that binds to an epitopewithin, or overlapping with, a peptide fragment of a human complementcomponent C5 protein. The peptide fragment can be a fragment of a humancomplement component C5 protein having the amino acid sequence depictedin any one of SEQ ID NOS:1-11. For example, the peptide fragment cancontain, or consist of, the following amino acid sequence:

(SEQ ID NO: 5) VIDHQGTKSSKCVRQKVEGSS.

As used herein, the term “crossblocking antibody” refers to an antibodythat lowers the amount of binding of anti-C5 antibody to an epitope on acomplement component C5 protein relative to the amount of binding of theanti-C5 antibody to the epitope in the absence of the antibody. Suitablemethods for determining whether a first antibody crossblocks binding ofa second antibody to an epitope are known in the art. For example,crossblocking antibodies can be identified by comparing the binding ofthe 5G1.1 anti-C5 monoclonal antibody (produced by the hybridoma cellline ATCC designation HB-11625; see U.S. Pat. No. 6,355,245) in thepresence and absence of a test antibody. Decreased binding of the 5G1.1antibody in the presence of the test antibody as compared to binding ofthe 5G1.1 antibody in the absence of the test antibody indicates thetest antibody is a crossblocking antibody.

Methods for identifying the epitope to which a particular antibody(e.g., an anti-C5 antibody) binds are also known in the art. Forexample, the binding epitope of an anti-C5 antibody can be identified bymeasuring the binding of the antibody to several (e.g., three, four,five, six, seven, eight, nine, 10, 15, 20, or 30 or more) overlappingpeptide fragments of a complement component C5 protein (e.g., severaloverlapping fragments of a protein having the amino acid sequencedepicted in any one of SEQ ID NOs:1-11). Each of the differentoverlapping peptides is then bound to a unique address on a solidsupport, e.g., separate wells of a multi-well assay plate. Next, theanti-C5 antibody is interrogated by contacting it to each of thepeptides in the assay plate for an amount of time and under conditionsthat allow for the antibody to bind to its epitope. Unbound anti-C5antibody is removed by washing each of the wells. Next, adetectably-labeled secondary antibody that binds to the anti-C5antibody, if present in a well of the plate, is contacted to each of thewells, and unbound secondary antibody is removed by washing steps. Thepresence or amount of the detectable signal produced by thedetectably-labeled secondary antibody in a well is an indication thatthe anti-C5 antibody binds to the particular peptide fragment associatedwith the well. See, e.g., Harlow and Lane (supra), Benny K. C. Lo(supra), and U.S. Patent Application Publication No. 20060153836, thedisclosure of which is incorporated by reference in its entirety. Aparticular epitope to which an antibody binds can also be identifiedusing BIAcore™ chromatographic techniques (see, e.g., PharmaciaBIAtechnology Handbook, “Epitope Mapping,” Section 6.3.2, (May 1994);and Johne et al. (1993) J Immunol Methods 160:191-8).

The anti-C5 antibodies described herein can have activity in blockingthe generation or activity of the C5a and/or C5b active fragments of acomplement component C5 protein (e.g., a human C5 protein). Through thisblocking effect, the anti-C5 antibodies inhibit, e.g., theproinflammatory effects of C5a and the generation of the C5b-9 membraneattack complex (MAC) at the surface of a cell. Anti-C5 antibodies thathave the ability to block the generation of C5a are described in, e.g.,Moongkarndi et al. (1982) Immunobiol 162:397 and Moongkarndi et al.(1983) Immunobiol 165:323.

Inhibition of complement component C5 can also reduce the cell-lysingability of complement in a subject's body fluids. Such reductions of thecell-lysing ability of complement present can be measured by methodswell known in the art such as, for example, by a conventional hemolyticassay such as the hemolysis assay described by Kabat and Mayer (eds),“Experimental Immunochemistry, 2^(nd) Edition,” 135-240, Springfield,Ill., CC Thomas (1961), pages 135-139, or a conventional variation ofthat assay such as the chicken erythrocyte hemolysis method as describedin, e.g., Hillmen et al. (2004) N Engl J Med 350(6):552.

In some embodiments, an anti-C5 antibody, or antigen-binding fragmentthereof, can reduce the ability of a C5 protein to bind to humancomplement component C3b (e.g., C3b present in an AP or CP C5 convertasecomplex) by greater than 50 (e.g., greater than 55, 60, 65, 70, 75, 80,85, 90, or 95 or more) %. In some embodiments, upon binding to a C5protein, the anti-C5 antibody or antigen-binding fragment thereof canreduce the ability of the C5 protein to bind to complement component C4b(e.g., C4b present in a CP C5 convertase) by greater than 50 (e.g.,greater than 55, 60, 65, 70, 75, 80, 85, 90, or 95 or more) %. Methodsfor determining whether an antibody can block the generation or activityof the C5a and/or C5b active fragments of a complement component C5protein, or binding to complement component C4b or C3b, are known in theart and described in, e.g., U.S. Pat. No. 6,355,245 and Wurzner et al.(1991) Complement Inflamm 8:328-340. (See also below.)

In some embodiments, an anti-C5 antibody binds to an amino-terminalregion of the alpha chain of a complement component C5 protein, but doesnot bind to free C5a. Epitopes for an anti-C5 antibody within theamino-terminal region of the alpha chain include, e.g., epitopes withinthe human sequence VIDHQGTKSSKCVRQKVEGSS (SEQ ID NO:5).

In some embodiments, the composition comprises, and/or the antibody is,eculizumab (Soliris®; Alexion Pharmaceuticals, Inc., Cheshire, Conn.) ora biologically-active fragment thereof. (See, e.g., Kaplan (2002) CurrOpin Investig Drugs 3(7):1017-23; Hill (2005) Clin Adv Hematol Oncol3(11):849-50; and Rother et al. (2007) Nature Biotechnology25(11):1256-1488.) The amino acid sequence of the light chain ofeculizumab is as follows:DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:12). Theamino acid sequence of the heavy chain of eculizumab is as follows:

(SEQ ID NO: 13) QVQLVQSGAEVKKPGASVKVSCKASGYIFSNYWIQWVRQAPGQGLEWMGEILPGSGSTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYEDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK.

As described herein and exemplified in the working examples, thefeatured aqueous solutions provide the anti-C5 antibody formulatedtherein with marked stability—both physical/chemical stability as wellas functional stability. For example, the formulations described hereinare capable of maintaining the structural integrity of an anti-C5antibody present at high concentrations in a solution. That is, ananti-C5 antibody in a featured aqueous buffer can remain predominantlymonomeric after storage for at least one month (e.g., at least twomonths, three months, four months, five months, six months, sevenmonths, eight months, nine months, 10 months, 11 months, 12 months, 13months, 14 months, 15 months, 16 months, 17 months, 18 months, 19months, 20 months, 21 months, 22 months, 23 months, 24 months, or more)at approximately 2° C. to 8° C. (e.g., storage at, e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, or 10° C.). As exemplified in the working examples describedherein, the inventors provide formulations suitable for maintaining ananti-C5 antibody at approximately 30 mg/mL or approximately 100 mg/mL inpredominantly monomeric form for up to two years at approximately 2° C.to 8° C. As used herein, an anti-C5 antibody formulated at a highconcentration in a featured aqueous solution is “predominantlymonomeric,” or in “predominantly monomeric form,” if the antibodypresent in the solution is at least 95 (e.g., at least 95.1, 95.2, 95.3,95.4, 95.5, 95.6, 95.7, 95.8, 95.9, 96, 96.1, 96.2, 96.3, 96.4, 96.5,96.6, 96.7, 96.8, 96.9, 97, 97.1, 97.2, 97.3, 97.4, 97.5, 97.6, 97.7,97.8, 97.9, 98, 98.1, 98.2, 98.3, 98.4, 98.5, 98.6, 98.7, 98.8, 98.9,99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, or 99.9 or greater)% monomeric, e.g., as determined using size exclusion chromatographyhigh performance liquid chromatography (SEC-HPLC). That is: less than 5(e.g., less than 4.9. 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9,3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5,2.4, 2.3, 2.2, 2.1, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1,0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1) % of the antibody in thesolution is oligomeric, aggregated, and/or fragmented. As used herein,antibody fragmentation refers to improperly assembled constituents ordegradation products of a whole antibody having a lower molecular weightthan the whole antibody. Such fragmentation forms include, but are notlimited to, a free monomeric heavy chain polypeptide, a dimeric heavychain polypeptide (e.g., disulfide-linked heavy chain polypeptide), adimeric heavy chain polypeptide bound to one light chain polypeptide, amonomeric heavy chain polypeptide bound to one light chain polypeptide,or further degradation product(s) or fragment(s) of a light chain orheavy chain polypeptide. In some embodiments, less than 2 (e.g., lessthan 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6,0.5, 0.4, 0.3, 0.2, or 0.1) % of the antibody is aggregated afterstorage for at least one month (e.g., at least two months, three months,four months, five months, six months, seven months, eight months, ninemonths, 10 months, 11 months, 12 months, 13 months, 14 months, 15months, 16 months, 17 months, 18 months, 19 months, 20 months, 21months, 22 months, 23 months, 24 months, or more) at 2° C. to 8° C. Insome embodiments, less than 1 (e.g., less than 0.9, 0.8, 0.7, 0.6, 0.5,0.4, 0.3, 0.2, or 0.1) % of the antibody is fragmented after storage forat least one month (e.g., at least two months, three months, fourmonths, five months, six months, seven months, eight months, ninemonths, 10 months, 11 months, 12 months, 13 months, 14 months, 15months, 16 months, 17 months, 18 months, 19 months, 20 months, 21months, 22 months, 23 months, 24 months, or more) at 2° C. to 8° C.Methods for determining the amount of monomeric antibody, as well as theamount of oligomeric, aggregated, or fragmented forms of the anti-C5antibody present in solution are described herein and exemplified in theworking examples. For example, a skilled artisan can determine thepercentage of whole, fragmented, unfolded intermediates, and/oraggregated antibody species present in a given solution using, e.g.,size exclusion chromatography high-performance liquid chromatography(SEC-HPLC), static light scattering (SLS), Fourier transform infraredspectroscopy (FTIR), circular dichroism (CD), urea-induced proteinunfolding techniques, intrinsic tryptophan fluorescence, non-reducingsodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE),and differential scanning calorimetry (DSC). In the working examplesdescribed herein, the inventors exemplify the use of, among others,SEC-HPLC and SDS-PAGE to determine the physical state of the anti-C5antibodies in solution.

In some embodiments, the formulation conditions described herein arecapable of maintaining the anti-C5 antibody in at least 95 (e.g., atleast 95.1, 95.2, 95.3, 95.4, 95.5, 95.6, 95.7, 95.8, 95.9, 96, 96.1,96.2, 96.3, 96.4, 96.5, 96.6, 96.7, 96.8, 96.9, 97, 97.1, 97.2, 97.3,97.4, 97.5, 97.6, 97.7, 97.8, 97.9, 98, 98.1, 98.2, 98.3, 98.4, 98.5,98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7,99.8, or 99.9 or greater) % monomeric form when stored for at least onemonth (e.g., at least two months, three months, four months, fivemonths, six months, seven months, eight months, nine months, 10 months,11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17months, 18 months, 19 months, 20 months, 21 months, 22 months, 23months, 24 months, or more) at approximately −20° C. (e.g., −20±5° C.).The percentage of monomeric form of the antibody in solution can bedetermined using SEC-HPLC. That is: less than 5 (e.g., less than 4.9.4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5,3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2,1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5,0.4, 0.3, 0.2, or 0.1) % of the antibody in the solution can becomeoligomeric, aggregated, and/or fragmented, when the aqueous solution isstored for at least one month at −20° C. As described above, in someembodiments, less than 2 (e.g., less than 1.9, 1.8, 1.7, 1.6, 1.5, 1.4,1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1) % ofthe antibody is aggregated after storage for at least one month (e.g.,at least two months, three months, four months, five months, six months,seven months, eight months, nine months, 10 months, 11 months, 12months, 13 months, 14 months, 15 months, 16 months, 17 months, 18months, 19 months, 20 months, 21 months, 22 months, 23 months, 24months, or more) at approximately −20° C. In some embodiments, less than1 (e.g., less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1) % ofthe antibody is fragmented after storage for at least one month (e.g.,at least two months, three months, four months, five months, six months,seven months, eight months, nine months, 10 months, 11 months, 12months, 13 months, 14 months, 15 months, 16 months, 17 months, 18months, 19 months, 20 months, 21 months, 22 months, 23 months, 24months, or more) at −20° C.

As described herein and exemplified in the working examples, the anti-C5antibody containing solutions featured herein can retain at least 90(e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or even 100) % of theirbiological/functional activity (e.g., ability to bind to human C5) afterstorage for at least one month (e.g., at least two months, three months,four months, five months, six months, seven months, eight months, ninemonths, 10 months, 11 months, 12 months, 13 months, 14 months, 15months, 16 months, 17 months, 18 months, 19 months, 20 months, 21months, 22 months, 23 months, 24 months, or more) at 2° C. to 8° C.Antibody present in a featured solution can retain, in some embodiments,at least 90 (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or even 100) % ofits activity to inhibit hemolysis after storage for at least one month(e.g., at least two months, three months, four months, five months, sixmonths, seven months, eight months, nine months, 10 months, 11 months,12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18months, 19 months, 20 months, 21 months, 22 months, 23 months, 24months, or more) at 2° C. to 8° C. Suitable hemolytic assay methods fordetermining whether an antibody in a featured solution retains itsactivity are described herein and known in the art, e.g., in vitrohemolytic assays using avian or porcine erythrocytes. Suitable methodsfor evaluating the ability of an antibody preparation to bind to humancomplement component C5 are known in the art and described herein.

In some embodiments, any of the aqueous solutions described herein cancontain one or more common excipients and/or additives such as bufferingagents, sugars or saccharides, salts, and surfactants. Additionally oralternatively, the solutions can further contain one or moresolubilizers, diluents, binders, stabilizers, salts, lipophilicsolvents, amino acids, chelators, or preservatives.

The solutions described herein can also include a buffering orpH-adjusting agent. In some embodiments, any of the aqueous solutionsdescribed herein can have, or can be adjusted to have, a neutral pH. Asused herein, “neutral pH” is a pH that is between, and inclusive of, 7and 8. Accordingly, as used herein neutral pH is inclusive of particularpH values such as 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and8.0. In some embodiments, neutral pH is at least pH 7 (e.g., at least pH7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.7 or 7.9), but less than pH 8(e.g., less than pH 7.9, 7.8, 7.7, 7.6, 7.5, 7.4, 7.3, 7.2, or 7.1).That is, in some embodiments neutral pH can be, e.g., at least pH 7, butless than pH 7.5. In some embodiments, neutral pH can be between pH 7and pH 7.5. In some embodiments, neutral pH can be between pH 7 and pH7.2. In some embodiments, neutral pH can be, e.g., pH 7. One of skill inthe art will also appreciate that human blood (such as human blood froma healthy patient) has a neutral pH as defined herein, e.g., the pH ofhuman blood is approximately pH 7.35 to pH 7.45. See, e.g., Boron andBoulpaep (2003) “Medical physiology: a cellular and molecular approach,”W.B. Saunders, New York (ISBN:0721632564). In some embodiments, the pHof a highly-concentrated antibody solution described herein is betweenapproximately 6.4 and 7.5, inclusive (e.g., approximately 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, or 7.7).

Buffering agents useful in the aqueous solutions described hereininclude, e.g., salts of citric acid, ascorbic acid, gluconic acid,carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalicacid. In some embodiments, the buffer is a Tris-based or phosphatebuffer.

In some embodiments, the aqueous solutions described herein can includeone or more amino acids, which can, among other things, providebuffering capacity. Suitable amino acids for use in the solutionsfeatured herein include, e.g., histidine, glycine, and serine. In someembodiments, the featured solutions do not include a free amino acid asa buffering agent. In some embodiments, the featured solutions includebut one free amino acid (e.g., histidine) as a buffering agent. In someembodiments, the featured solutions can include two or more (e.g., two,three, four, five, six, or seven or more) different amino acids asbuffering agents, e.g., serine and histidine.

The buffering agents are generally used at concentrations betweenapproximately 1 mM and 200 mM, depending, in part, on the bufferingcapacity required. In some embodiments, an aqueous solution describedherein can include a buffering agent at a concentration of less than, orapproximately, 300 (e.g., less than, or approximately, 290, 280, 270,260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130,120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, or 10) mM. Insome embodiments, an aqueous solution described herein contains abuffering agent at a concentration of at least 10 (e.g., at least 15,20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or300 or more) mM. In some embodiments, an aqueous solution describedherein can include a buffering agent at a concentration of between about10 mM to 50 mM, 15 mM to 50 mM, 20 mM to 50 mM, 25 mM to 50 mM, 30 mM to50 mM, 40 mM to 50 mM, 10 mM to 100 mM, 15 mM to 100 mM, 20 mM to 100mM, 25 mM to 100 mM, 30 mM to 100 mM, 40 mM to 100 mM, 10 mM to 150 mM,15 mM to 150 mM, 20 mM to 150 mM, 25 mM to 150 mM, 30 mM to 150 mM, 40mM to 150 mM, 50 mM to 100 mM, 60 mM to 100 mM, 70 mM to 100 mM, 80 mMto 100 mM, 50 mM to 150 mM, 60 mM to 150 mM, 70 mM to 150 mM, 80 mM to150 mM, 90 mM to 150 mM, 100 mM to 150 mM, 10 mM to 200 mM, 15 mM to 200mM, 20 mM to 200 mM, 25 mM to 200 mM, 30 mM to 200 mM, 40 mM to 200 mM,50 mM to 200 mM, 60 mM to 200 mM, 70 mM to 200 mM, 80 mM to 200 mM, 90mM to 200 mM, 100 mM to 200 mM, 150 mM to 200 mM, 10 mM to 250 mM, 15 mMto 250 mM, 20 mM to 250 mM, 25 mM to 250 mM, 30 mM to 250 mM, 40 mM to250 mM, 50 mM to 250 mM, 60 mM to 250 mM, 70 mM to 250 mM, 80 mM to 250mM, 90 mM to 250 mM, 100 mM to 250 mM, 150 mM to 250 mM, or 200 mM to250 mM. It is understood that in embodiments where a featured solutioncontains two or more (e.g., at least two, three, four, five, six, seven,eight, nine, or 10 or more) different buffering agents, each of the twoor more buffering agents can independently be present at, e.g., one ofthe above described concentrations.

In some embodiments, any of the aqueous solutions described herein cancontain a salt, e.g., sodium chloride, potassium chloride, or magnesiumchloride. In some embodiments, an aqueous solution described hereincontains a salt at a concentration of at least 10 (e.g., at least 15,20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or300 or more) mM. In some embodiments, an aqueous solution describedherein can include a salt at a concentration of less than, orapproximately, 200 (e.g., less than, or approximately, 190, 180, 170,160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 25, 20,15, or 10) mM. In some embodiments, an aqueous solution described hereincan include a salt at a concentration of between about 10 mM to 50 mM,15 mM to 50 mM, 20 mM to 50 mM, 25 mM to 50 mM, 30 mM to 50 mM, 40 mM to50 mM, 10 mM to 100 mM, 15 mM to 100 mM, 20 mM to 100 mM, 25 mM to 100mM, 30 mM to 100 mM, 40 mM to 100 mM, 10 mM to 150 mM, 15 mM to 150 mM,20 mM to 150 mM, 25 mM to 150 mM, 30 mM to 150 mM, 40 mM to 150 mM, 50mM to 100 mM, 60 mM to 100 mM, 70 mM to 100 mM, 80 mM to 100 mM, 50 mMto 150 mM, 60 mM to 150 mM, 70 mM to 150 mM, 80 mM to 150 mM, 90 mM to150 mM, 100 mM to 150 mM, 10 mM to 200 mM, 15 mM to 200 mM, 20 mM to 200mM, 25 mM to 200 mM, 30 mM to 200 mM, 40 mM to 200 mM, 50 mM to 200 mM,60 mM to 200 mM, 70 mM to 200 mM, 80 mM to 200 mM, 90 mM to 200 mM, 100mM to 200 mM, 150 mM to 200 mM, 10 mM to 250 mM, 15 mM to 250 mM, 20 mMto 250 mM, 25 mM to 250 mM, 30 mM to 250 mM, 40 mM to 250 mM, 50 mM to250 mM, 60 mM to 250 mM, 70 mM to 250 mM, 80 mM to 250 mM, 90 mM to 250mM, 100 mM to 250 mM, 150 mM to 250 mM, or 200 mM to 250 mM. It isunderstood that in embodiments where a featured solution contains two ormore (e.g., at least two, three, four, five, six, seven, eight, nine, or10 or more) different salts, each of the two or more salts canindependently be present at, e.g., one of the above describedconcentrations.

In some embodiments, any of the aqueous solutions described herein cancontain a carbohydrate excipient. Suitable carbohydrate excipients aredescribed in, e.g., Katakam and Banga (1995) J Pharm Pharmacol47(2):103-107; Andya et al. (2003) AAPS PharmSci 5(2): Article 10; andShire (2009) “Current Trends in Monoclonal Antibody Development andManufacturing,” Volume 11, Springer, 354 pages. Carbohydrate excipientssuitable for use in the solutions described herein include, withoutlimitation, monosaccharides such as fructose, maltose, galactose,glucose, D-mannose, and sorbose; disaccharides such as lactose, sucrose,trehalose, and cellobiose; polysaccharides such as maltodextrins,dextrans, and starches; and sugar alcohols such as mannitol, xylitol,maltitol, lactitol, and sorbitol. In some embodiments, a carbohydrateexcipient is present in a solution featured herein at a concentration ofat least, or approximately, 0.5 (e.g., at least, or approximately, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.25, 3.5, 3.75, 4, 4.25,4.5, 4.75, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or more) %. Inembodiments where a featured solution contains two or more (e.g., atleast two, three, four, five, six, seven, eight, nine, or 10 or more)different carbohydrate excipients (e.g., sorbitol and mannitol), eachexcipient can, independently, be present at any of the above-describedconcentrations.

In some embodiments, an aqueous solution described herein can contain asurfactant such as an anionic, cationic, or nonionic surfactant.Pharmaceutically-acceptable surfactants include, without limitation:polysorbates: Triton™ (e.g., Triton™ 20 or Triton™ 80), sodium octylglycoside; lauryl-, myristyl-, linoleyl-, or stearylsulfobetaine;lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-,myristyl-, or cetylbetaine; lauroamidopropyl-, cocamidopropyl-,linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-betaine (e.g. lauroamidopropyl); myristamidopropyl-,palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methylcocoyl-, or disodium methyl oleyl-taurate; and polyethyl glycol,polypropyl glycol, and copolymers of ethylene and propylene glycol. Insome embodiments, the aqueous solutions described herein contain asurfactant (e.g., any of the pharmaceutically-acceptable surfactantsdescribed herein or known in the art) at a concentration of at least, orapproximately, 0.001 (e.g., at least, or approximately, 0.002, 0.003,0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05,0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17,0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29,0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41,0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5 or more) %. Insome embodiments, an aqueous solution described herein contains no morethan 0.2 (e.g., no more than 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13,0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01,0.009, 0.008, 0.007, 0.006, 0.005, 0.004, 0.003, 0.002, or 0.001) % of apharmaceutically-acceptable surfactant.

In some embodiments, an aqueous solution described herein can beformulated to comprise the following elements: 20 mM histidine, 50 mMserine, 3% sorbitol, and 1.5% mannitol. In some embodiments, thissolution is formulated at pH 7. In some embodiments, the aqueoussolution can consist of the foregoing elements along with an anti-C5antibody (e.g., any one of the anti-C5 antibodies described herein) atany of the high concentrations described herein.

In some embodiments, an aqueous solution described herein can comprise,or consist of: (i) an anti-C5 antibody (e.g., eculizumab) at aconcentration of at least, or approximately, 80 (e.g., at least, orapproximately, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140,145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220,230, 240, or 250 or more) mg/mL; (ii) at least, or approximately, 10(e.g., at least, or approximately, 15 or 20 or more) mM histidine; (ii)at least, or approximately, 10 (e.g., at least, or approximately, 15,20, 25, 30, 35, 40, 45, or 50 or more) mM serine; (iv) at least, orapproximately, 1 (e.g., at least, or approximately, 1.5, 2, 2.5, or 3 ormore) % sorbitol; and (v) at least 0.5 (e.g., at least, orapproximately, 0.75, 1, 1.25, or 1.5 or more) % mannitol, wherein thesolution is formulated at neutral pH (e.g., a pH of approximately 7).

In some embodiments, an aqueous solution described herein can comprise,or consist of: (i) eculizumab at a concentration of at least, orapproximately 100 mg/mL; (ii) at least, or approximately, 20 mMhistidine; (ii) at least, or approximately, 50 mM serine; (iv) at least,or approximately, 3% sorbitol; and (v) at least, or approximately, 1.5%mannitol, wherein the solution is formulated at a pH of approximately 7.

In some embodiments, an aqueous solution described herein can beformulated to comprise the following elements: 10 mM histidine HCl, 10%alpha-trehalose dihydrate, and 0.01% polysorbate 20. In someembodiments, this solution is formulated at pH 7. In some embodiments,the aqueous solution can consist of the foregoing elements along with ananti-C5 antibody (e.g., any one of the anti-C5 antibodies describedherein) at any of the high concentrations described herein.

In some embodiments, the aqueous solutions described herein do notcontain the following elements at the recited concentrations and pH: 20mM histidine; 50 nM glycine; 3% (w/v) sorbitol; 1.5% (w/v) mannitol;0.001% to 0.02% Tween 80; and a pH of 6 to 8. In some embodiments, anaqueous solution described herein does not contain trehalose (e.g.,alpha-trehalose). In some embodiments, an aqueous solution describedherein is not a phosphate-based buffer (e.g., phosphate bufferedsaline). For example, in some embodiments, the solution does not containsodium phosphate. In some embodiments, the aqueous solutions describedherein do not contain the following elements at the recitedconcentrations: 10 mM sodium phosphate, 150 mM sodium chloride, 0.001%to 0.02% Tween 80, and at a pH of 6 to 8.

In some embodiments, any of the aqueous solutions described herein areisotonic with respect to human blood. In some embodiments, a solutiondescribed herein has an osmotic pressure of between approximately 270mOsm/kg and 328 mOsm/kg, e.g., approximately, 270 mOsm/kg, 275 mOsm/kg,280 mOsm/kg, 285 mOsm/kg, 290 mOsm/kg, 295 mOsm/kg, 300 mOsm/kg, 305mOsm/kg, 310 mOsm/kg, 315 mOsm/kg, 320 mOsm/kg, 325 mOsm/kg, or 328mOsm/kg. In some embodiments, a solution described herein has an osmoticpressure that is at least or greater than 250 (e.g., at least, orgreater than, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305,310, 315, 320, 325, or 327) mOsm/kg, but not more than, or less than,350 (e.g., not more than, or less than, 345, 340, 335, 327, 325, 320,315, 310, 305, or 300) mOsm/kg. In some embodiments, the solutionsdescribed herein can contain, or be formulated with, one or moretonicity agents useful for maintaining or modulating the osmoticpressure of a solution. For example, a solution described herein cancontain one or more amino acids, certain pharmaceutically-acceptablesalts, or sugars.

The aqueous solutions described herein can be sterile,pharmaceutical-grade compositions, e.g., for administration to a subjectfor the treatment or prevention of a complement-associated disorder. Thecompositions can be formulated according to standard methods.Pharmaceutical formulation is a well-established art, and is furtherdescribed in, e.g., Gennaro (2000) “Remington: The Science and Practiceof Pharmacy,” 20^(th) Edition, Lippincott, Williams & Wilkins (ISBN:0683306472); Ansel et al. (1999) “Pharmaceutical Dosage Forms and DrugDelivery Systems,” 7^(th) Edition, Lippincott Williams & WilkinsPublishers (ISBN: 0683305727); and Kibbe (2000) “Handbook ofPharmaceutical Excipients American Pharmaceutical Association,” 3^(rd)Edition (ISBN: 091733096X). Suitable formulation methods for the highconcentration antibody solutions described herein are exemplified in theworking examples.

In some embodiments, a high concentration antibody solution describedherein can be formulated for delivery to the eye. As used herein, theterm “eye” refers to any and all anatomical tissues and structuresassociated with an eye. In some embodiments, an aqueous solutiondescribed herein can be administered locally, for example, by way oftopical application or intravitreal injection. For example, in someembodiments, solution can be formulated for administration by way of aneye dropper.

In some embodiments, a sterile, aqueous solution contains, e.g.,additional ingredients such as, but not limited to, preservatives,buffers, tonicity agents, antioxidants and stabilizers, nonionic wettingor clarifying agents, and viscosity-increasing agents. Suitablepreservatives for use in such a solution include benzalkonium chloride,benzethonium chloride, chlorobutanol, thimerosal and the like. Suitablebuffers include, e.g., boric acid, sodium and potassium bicarbonate,sodium and potassium borates, sodium and potassium carbonate, sodiumacetate, and sodium biphosphate, in amounts sufficient to maintain thepH at between about pH 6 and pH 8 (see above for suitable pH ranges).Suitable tonicity agents are dextran 40, dextran 70, dextrose, glycerin,potassium chloride, propylene glycol, and sodium chloride.

Suitable antioxidants and stabilizers include sodium bisulfite, sodiummetabisulfite, sodium thiosulfite, and thiourea. Suitable wetting andclarifying agents include polysorbate 80, polysorbate 20, poloxamer 282and tyloxapol. Suitable viscosity-increasing agents include dextran 40,dextran 70, gelatin, glycerin, hydroxyethylcellulose,hydroxmethylpropylcellulose, lanolin, methylcellulose, petrolatum,polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, andcarboxymethylcellulose.

In some embodiments, a highly-concentrated antibody solution describedherein can be formulated for administration to the eye, e.g., topicaladministration to the eye of the subject in need of treatment (e.g., asubject afflicted with AMD). For example, in some embodiments, ahighly-concentrated antibody solution described herein can be formulatedas an eye drop. In addition, a highly-concentrated antibody solutiondescribed herein can be formulated for use with any of a variety ofdevices developed for introducing therapeutic compounds into the vitrealcavity of the eye. For example, U.S. patent application publication no.20020026176 describes a pharmaceutical-containing plug that can beinserted through the sclera such that it projects into the vitreouscavity to deliver the pharmaceutical agent into the vitreous cavity. Inanother example, U.S. Pat. No. 5,443,505 describes an implantable devicefor introduction into a suprachoroidal space or an avascular region forsustained release of drug into the interior of the eye. Additionalmethods and devices (e.g., a transscleral patch and delivery via contactlenses) for delivery of a therapeutic agent to the eye are described in,e.g., Ambati and Adamis (2002) Prog Retin Eye Res 21(2):145-151; Rantaand Urtti (2006) Adv Drug Delivery Rev 58(11):1164-1181; Barocas andBalachandran (2008) Expert Opin Drug Delivery 5(1):1-10(10); Gulsen andChauhan (2004) Invest Opthalmol Vis Sci 45:2342-2347; Kim et al. (2007)Ophthalmic Res 39:244-254; and PCT publication no. WO 04/073551, thedisclosures of which are incorporated herein by reference in theirentirety.

In some embodiments, an antibody or antigen-binding fragment describedherein can be formulated in a composition suitable for intrapulmonaryadministration (e.g., for administration via nebulization; see below) toa mammal such as a human. Methods for preparing such compositions arewell known in the art and described in, e.g., U.S. patent applicationpublication no. 20080202513; U.S. Pat. Nos. 7,112,341 and 6,019,968; andPCT application publication nos. WO 00/061178 and WO 06/122257, thedisclosures of each of which are incorporated herein by reference intheir entirety. Dry powder inhaler formulations and suitable systems foradministration of the formulations are described in, e.g., U.S. patentapplication publication no. 20070235029, PCT Publication No. WO00/69887; and U.S. Pat. No. 5,997,848.

Pulmonary drug delivery may be achieved by inhalation, andadministration by inhalation herein may be oral and/or nasal. Examplesof pharmaceutical devices for pulmonary delivery include metered doseinhalers and nebulizers. For example, an antibody or antigen-bindingfragment thereof can be administered to the lungs of a subject by way ofa nebulizer. Nebulizers use compressed air to deliver a compound as aliquefied aerosol or mist. A nebulizer can be, e.g., a jet nebulizer(e.g., air or liquid jet nebulizers) or an ultrasonic nebulizer.Additional devices and intrapulmonary administration methods are setforth in, e.g., U.S. Patent Application Publication Nos. 20050271660 and20090110679, the disclosures of each of which are incorporated herein byreference in their entirety.

In some embodiments, the solutions provided herein are present in unitdosage form, which can be particularly suitable for self-administration.A formulated product of the present disclosure can be included within acontainer, typically, for example, a vial, cartridge, prefilled syringeor disposable pen. A doser such as the doser device described in U.S.Pat. No. 6,302,855 may also be used, for example, with an injectionsystem of the present disclosure.

An injection system of the present disclosure may employ a delivery penas described in U.S. Pat. No. 5,308,341. Pen devices, most commonly usedfor self-delivery of insulin to patients with diabetes, are well knownin the art. Such devices can comprise at least one injection needle(e.g., a 31 gauge needle of about 5 to 8 mm in length), are typicallypre-filled with one or more therapeutic unit doses of a therapeuticsolution, and are useful for rapidly delivering the solution to asubject with as little pain as possible.

One medication delivery pen includes a vial holder into which a vial ofinsulin or other medication may be received. The vial holder is anelongate generally tubular structure with proximal and distal ends. Thedistal end of the vial holder includes mounting means for engaging adouble-ended needle cannula. The proximal end also includes mountingmeans for engaging a pen body which includes a driver and dose settingapparatus. A disposable medication (e.g., a high concentration solutionof an anti-C5 antibody) containing vial for use with the prior art vialholder includes a distal end having a pierceable elastomeric septum thatcan be pierced by one end of a double-ended needle cannula. The proximalend of this vial includes a stopper slidably disposed in fluid tightengagement with the cylindrical wall of the vial. This medicationdelivery pen is used by inserting the vial of medication into the vialholder. A pen body then is connected to the proximal end of the vialholder. The pen body includes a dose setting apparatus for designating adose of medication to be delivered by the pen and a driving apparatusfor urging the stopper of the vial distally for a distance correspondingto the selected dose. The user of the pen mounts a double-ended needlecannula to the distal end of the vial holder such that the proximalpoint of the needle cannula pierces the septum on the vial. The patientthen selects a dose and operates the pen to urge the stopper distally todeliver the selected dose. The dose selecting apparatus returns to zeroupon injection of the selected dose. The patient then removes anddiscards the needle cannula, and keeps the prior art medication deliverypen in a convenient location for the next required medicationadministration. The medication in the vial will become exhausted afterseveral such administrations of medication. The patient then separatesthe vial holder from the pen body. The empty vial may then be removedand discarded. A new vial can be inserted into the vial holder, and thevial holder and pen body can be reassembled and used as explained above.Accordingly, a medication delivery pen generally has a drive mechanismfor accurate dosing and ease of use.

A dosage mechanism such as a rotatable knob allows the user toaccurately adjust the amount of medication that will be injected by thepen from a prepackaged vial of medication. To inject the dose ofmedication, the user inserts the needle under the skin and depresses theknob once as far as it will depress. The pen may be an entirelymechanical device or it may be combined with electronic circuitry toaccurately set and/or indicate the dosage of medication that is injectedinto the user. See U.S. Pat. No. 6,192,891.

In some embodiments, the needle of the pen device is disposable and thekits include one or more disposable replacement needles. Pen devicessuitable for delivery of the any one of the presently featured antibodysolutions are also described in, e.g., U.S. Pat. Nos. 6,277,099;6,200,296; and 6,146,361, the disclosures of each of which areincorporated herein by reference in their entirety. A microneedle-basedpen device is described in, e.g., U.S. Pat. No. 7,556,615, thedisclosure of which is incorporated herein by reference in its entirety.See also the Precision Pen Injector (PPI) device, Molly™, manufacturedby Scandinavian Health Ltd.

The present disclosure also presents controlled-release orextended-release formulations suitable for chronic and/orself-administration of a medication. The various formulations can beadministered to a patient in need of treatment with the medication as abolus or by continuous infusion over a period of time.

In some embodiments, a high concentration anti-C5 antibody solutiondescribed herein is formulated for sustained-release, extended-release,timed-release, controlled-release, or continuous-release administration.In some embodiments, depot formulations are used to administer theantibody to the subject in need thereof. In this method, the antibody isformulated with one or more carriers providing a gradual release ofactive agent over a period of a number of hours or days. Suchformulations are often based upon a degrading matrix which graduallydisperses in the body to release the active agent.

In some embodiments, a highly-concentrated antibody solution describedherein can be formulated with one or more additional active agentsuseful for treating or preventing a complement-associated disorder(e.g., an AP-associated disorder or a CP-associated disorder) in asubject. Additional agents for treating a complement-associated disorderin a subject will vary depending on the particular disorder beingtreated, but can include, without limitation, an antihypertensive (e.g.,an angiotensin-converting enzyme inhibitor) [for use in treating, e.g.,HELLP syndrome], an anticoagulant, a corticosteroid (e.g., prednisone),or an immunosuppressive agent (e.g., vincristine or cyclosporine A).Examples of anticoagulants include, e.g., warfarin (Coumadin), aspirin,heparin, phenindione, fondaparinux, idraparinux, and thrombin inhibitors(e.g., argatroban, lepirudin, bivalirudin, or dabigatran). An anti-C5antibody described herein can also be formulated with a fibrinolyticagent (e.g., ancrod, ε-aminocaproic acid, antiplasmin-a₁, prostacyclin,and defibrotide) for the treatment of a complement-associated disorder.In some embodiments, an anti-C5 antibody can be formulated with alipid-lowering agent such as an inhibitor of hydroxymethylglutaryl CoAreductase. In some embodiments, an anti-C5 antibody can be formulatedwith, or for use with, an anti-CD20 agent such as rituximab (Rituxan™;Biogen Idec, Cambridge, Mass.). In some embodiments, e.g., for thetreatment of RA, an anti-C5 antibody can be formulated with one or bothof infliximab (Remicade®; Centocor, Inc.) and methotrexate (Rheumatrex®,Trexall®). In some embodiments, an anti-C5 antibody described herein canbe formulated with a non-steroidal anti-inflammatory drug (NSAID). Manydifferent NSAIDS are available, some over the counter includingibuprofen (Advil®, Motrin®, Nuprin®) and naproxen (Alleve®) and manyothers are available by prescription including meloxicam (Mobic®),etodolac (Lodine®), nabumetone (Relafen®), sulindac (Clinoril®),tolementin (Tolectin®), choline magnesium salicylate (Trilasate®),diclofenac (Cataflam®, Voltaren®, Arthrotec®), Diflusinal (Dolobid®),indomethicin (Indocin®), ketoprofen (Orudis®, Oruvail®), oxaprozin(Daypro®), and piroxicam (Feldene®). In some embodiments a C5-bindingpolypeptide can be formulated for use with an anti-hypertensive, ananti-seizure agent (e.g., magnesium sulfate), or an anti-thromboticagent. Anti-hypertensives include, e.g., labetalol, hydralazine,nifedipine, calcium channel antagonists, nitroglycerin, or sodiumnitroprussiate. (See, e.g., Mihu et al. (2007) J Gastrointestin LiverDis 16(4):419-424.) Anti-thrombotic agents include, e.g., heparin,antithrombin, prostacyclin, or low dose aspirin.

In some embodiments, a highly-concentrated antibody solution describedherein can be formulated for administration with one or more additionaltherapeutic agents for use in treating a complement-associated disorderof the eye. Such additional therapeutic agents can be, e.g., bevacizumabor the Fab fragment of bevacizumab or ranibizumab, both sold by RochePharmaceuticals, Inc., and pegaptanib sodium (Mucogen®; Pfizer, Inc.).Such a kit can also, optionally, include instructions for administeringthe anti-C5 antibody to a subject.

In some embodiments, a highly-concentrated antibody solution describedherein can be formulated with one or more additional therapeutic agentsfor use in treating a complement-associated pulmonary disorder such as,but not limited to, asthma, chronic obstructive pulmonary disease, acuterespiratory distress syndrome, pulmonary fibrosis, α-1 anti-trypsindeficiency, emphysema, bronchiectasis, bronchiolitis obliterans,sarcoidosis, a collagen vascular disorder, and bronchitis. Suchadditional therapeutic agents include, e.g., sympathomimetics (e.g.,albuterol), antibiotics, deoxyribonucleases (e.g., Pulmozyme®),anticholinergic drugs, anti-IgE inhibitors (e.g., anti-IgE antibodies),and corticosteroids.

In some embodiments, a highly-concentrated antibody solution describedherein can be formulated for administration to a subject along withintravenous gamma globulin therapy (IVIG), plasmapheresis, plasmareplacement, or plasma exchange. In some embodiments, an anti-C5antibody can be formulated for use before, during, or after a kidneytransplant.

When a highly-concentrated antibody solution described herein is to beused in combination with a second active agent, the agents can beformulated separately or together. For example, the respectivepharmaceutical compositions can be mixed, e.g., just prior toadministration, and administered together or can be administeredseparately, e.g., at the same or different times (see below).

Methods for Preparing the Highly-Concentrated Antibody Solutions

The disclosure also provides exemplary methods for preparing ahighly-concentrated antibody solution containing more than 100 mg/mL ofan anti-C5 antibody. For example, as described herein and exemplified inthe working examples, the inventors have identified improved methods forconcentrating an anti-C5 antibody solution that results not only in ahigher recovery of antibody from the process, but also a moreconcentrated final solution. That is, under this method the anti-C5antibody eculizumab can be concentrated in solution up to 224 mg/mL withan 85% recovery of the antibody starting material.

The method requires a first aqueous solution comprising an anti-C5antibody, the first aqueous solution having a first formulation andcomprising, preferably, no more than 50 mg/mL of the anti-C5 antibody.In some embodiments, the first aqueous solution having a firstformulation comprises no more than approximately 40 mg/mL of the anti-C5antibody. In some embodiments, the provided solution contains betweenabout 20 to about 50 (e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or49) mg/mL of the anti-C5 antibody. In some embodiments, the providedsolution contains less than 50 (e.g., less than 49, 48, 47, 46, 45, 44,43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, or 28) mg/mLof the anti-C5 antibody. The first formulation buffer can be, e.g., aphosphate-based buffer such as phosphate-buffered saline. Suitablephosphate-based buffers for use in these preparation methods are setforth in the working examples. In some embodiments, the first aqueoussolution is initially concentrated to around 30 mg/mL to 50 mg/mL of theanti-C5 antibody. For example, the first aqueous solution can beconcentrated to around 30 mg/mL to 40 mg/mL of the anti-C5 antibodyusing a tangential flow filter (TFF) or a stir cell. In someembodiments, the first aqueous solution is obtained by concentrating a“starting” solution having an anti-C5 antibody concentration of lessthan, or equal to, 15 (e.g., less than or equal to 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4, 3, 2 or even 1) mg/mL. The formulation of the startingsolution can be the same or can be different from the formulation of thefirst aqueous solution.

The method includes subjecting the first aqueous solution todiafiltration to thereby produce a second aqueous solution.Diafiltration is a membrane-based separation process useful forreducing, removing, or exchanging salts or other buffer components froma solution of interest. Diafiltration involves a buffering-exchangingprocess where a protein solution (e.g., the first solution containingthe anti-C5 antibody) is placed onto a filter having a specified poresize, wherein pressure applied to the solution upon the column forcescomponents of the solution that are smaller than the pores of the filterto pass through the filter. Higher molecular weight species, such as ananti-C5 antibody, that are unable to pass through the pores of thefilter are retained (retentate). By applying a volume of a second buffersolution into the retentate container during the diafiltration process,the lower molecular weight buffer components can be exchanged resultingin a retentate having a different formulation than that of the firstsolution. Typically, a volume of the second buffer equal to volume ofthe retentate is applied in each “round” of diafiltration. For example,5 mL of the first solution can placed on the filter and 5 mL of thesecond buffer is added to the first solution before or during theapplication of a pressure suitable to gently force the lower molecularweight components of the buffer (e.g., water, salts, etc.) through thefilter. In such an example, pressure would be applied until the initial10 mL volume was reduced to a 5 mL retentate. A second round ofdiafiltration could be performed wherein an additional 5 mL of thesecond buffer is applied to the retentate upon the filter. Pressure isapplied to the solution on the filter until the 10 mL volume is reducedagain to 5 mL. In some embodiments, diafiltration can involve the use ofpressure and/or tangential flow to force low molecular weight moleculesacross a flat sheet membrane. In some embodiments, the diafiltrationbuffer can be added continuously during the process to maintain aconstant retentate volume while the buffer is exchanged.

In some embodiments, one round of diafiltration will be performed. Insome embodiments, two or more (e.g., three, four, five, six, seven,eight, nine, or 10 or more) rounds of diafiltration are performed.Following the one or more rounds of diafiltration, the originalformulation of the first aqueous solution has been exchanged to a secondformulation, thus resulting in a second aqueous solution. In someembodiments, the second formulation comprises: at least 20 mM histidine;at least 50 mM serine; at least 2.5% (w/v) sorbitol; and at least 1.5%(w/v) mannitol. Exemplary second formulations are described herein andexemplified in the working examples.

The methods can also include, following the diafiltration step,concentrating the second aqueous solution to produce a concentratedantibody solution comprising greater than 100 mg/mL of the anti-C5antibody. In some embodiments, the concentration step is performed toproduce a concentrated antibody solution having greater than 125 (e.g.,130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195,200, 205, 210, 215, 220, or greater than 225) mg/mL. The concentrationstep can include, e.g., tangential flow filtration or a stir cell.

As described herein, the methods allow at least 90 (e.g., at least 91,92, 93, 94, 95, 96, 97, 98, 99, or even 100) % of the anti-C5 antibodypresent in the first aqueous solution to be recovered in the highconcentration aqueous solution.

Methods for Producing an Antibody

Suitable methods for producing an antibody, or antigen-binding fragmentsthereof, in accordance with the disclosure are known in the art (see,e.g., U.S. Pat. No. 6,355,245) and described herein. For example,monoclonal anti-C5 antibodies may be generated using complementcomponent C5-expressing cells, a C5 polypeptide, or an antigenicfragment of C5 polypeptide, as an immunogen, thus raising an immuneresponse in animals from which antibody-producing cells and in turnmonoclonal antibodies may be isolated. The sequence of such antibodiesmay be determined and the antibodies or variants thereof produced byrecombinant techniques. Recombinant techniques may be used to producechimeric, CDR-grafted, humanized and fully human antibodies based on thesequence of the monoclonal antibodies as well as polypeptides capable ofbinding to human complement component C5.

Moreover, antibodies derived from recombinant libraries (“phageantibodies”) may be selected using C5-expressing cells, or polypeptidesderived therefrom, as bait to isolate the antibodies or polypeptides onthe basis of target specificity. The production and isolation ofnon-human and chimeric anti-C5 antibodies are well within the purview ofthe skilled artisan.

Recombinant DNA technology can be used to modify one or morecharacteristics of the antibodies produced in non-human cells. Thus,chimeric antibodies can be constructed in order to decrease theimmunogenicity thereof in diagnostic or therapeutic applications.Moreover, immunogenicity can be minimized by humanizing the antibodiesby CDR grafting and, optionally, framework modification. See, U.S. Pat.Nos. 5,225,539 and 7,393,648, the contents of each of which areincorporated herein by reference.

Antibodies can be obtained from animal serum or, in the case ofmonoclonal antibodies or fragments thereof, produced in cell culture.Recombinant DNA technology can be used to produce the antibodiesaccording to established procedure, including procedures in bacterial orpreferably mammalian cell culture. The selected cell culture systempreferably secretes the antibody product.

In another embodiment, a process for the production of an antibodydisclosed herein includes culturing a host, e.g., E. coli or a mammaliancell, which has been transformed with a hybrid vector. The vectorincludes one or more expression cassettes containing a promoter operablylinked to a first DNA sequence encoding a signal peptide linked in theproper reading frame to a second DNA sequence encoding the antibodyprotein. The antibody protein is then collected and isolated.Optionally, the expression cassette may include a promoter operablylinked to polycistronic (e.g., bicistronic) DNA sequences encodingantibody proteins each individually operably linked to a signal peptidein the proper reading frame.

Multiplication of hybridoma cells or mammalian host cells in vitro iscarried out in suitable culture media, which include the customarystandard culture media (such as, for example Dulbecco's Modified EagleMedium (DMEM) or RPMI 1640 medium), optionally replenished by amammalian serum (e.g. fetal calf serum), or trace elements and growthsustaining supplements (e.g. feeder cells such as normal mouseperitoneal exudate cells, spleen cells, bone marrow macrophages,2-aminoethanol, insulin, transferrin, low density lipoprotein, oleicacid, or the like). Multiplication of host cells which are bacterialcells or yeast cells is likewise carried out in suitable culture mediaknown in the art. For example, for bacteria suitable culture mediainclude medium LE, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2×YT, orM9 Minimal Medium. For yeast, suitable culture media include medium YPD,YEPD, Minimal Medium, or Complete Minimal Dropout Medium.

In vitro production provides relatively pure antibody preparations andallows scale-up production to give large amounts of the desiredantibodies. Techniques for bacterial cell, yeast, plant, or mammaliancell cultivation are known in the art and include homogeneous suspensionculture (e.g. in an airlift reactor or in a continuous stirrer reactor),and immobilized or entrapped cell culture (e.g. in hollow fibers,microcapsules, on agarose microbeads or ceramic cartridges).

Large quantities of the desired antibodies can also be obtained bymultiplying mammalian cells in vivo. For this purpose, hybridoma cellsproducing the desired antibodies are injected into histocompatiblemammals to cause growth of antibody-producing tumors. Optionally, theanimals are primed with a hydrocarbon, especially mineral oils such aspristane (tetramethyl-pentadecane), prior to the injection. After one tothree weeks, the antibodies are isolated from the body fluids of thosemammals. For example, hybridoma cells obtained by fusion of suitablemyeloma cells with antibody-producing spleen cells from Balb/c mice, ortransfected cells derived from hybridoma cell line Sp2/0 that producethe desired antibodies are injected intraperitoneally into Balb/c miceoptionally pre-treated with pristane. After one to two weeks, asciticfluid is taken from the animals.

The foregoing, and other, techniques are discussed in, for example,Kohler and Milstein, (1975) Nature 256:495-497; U.S. Pat. No. 4,376,110;Harlow and Lane, Antibodies: a Laboratory Manual, (1988) Cold SpringHarbor, the disclosures of which are all incorporated herein byreference. Techniques for the preparation of recombinant antibodymolecules are described in the above references and also in, e.g.:WO97/08320; U.S. Pat. No. 5,427,908; U.S. Pat. No. 5,508,717; Smith(1985) Science 225:1315-1317; Parmley and Smith (1988) Gene 73:305-318;De La Cruz et al. (1988) J Biol Chem 263:4318-4322; U.S. Pat. No.5,403,484; U.S. Pat. No. 5,223,409; WO88/06630; WO92/15679; U.S. Pat.No. 5,780,279; U.S. Pat. No. 5,571,698; U.S. Pat. No. 6,040,136; Daviset al. (1999) Cancer Metastasis Rev 18(4):421-5; and Taylor et al.(1992) Nucleic Acids Res 20: 6287-6295; Tomizuka et al. (2000) Proc NatlAcad Sci USA 97(2): 722-727, the contents of each of which areincorporated herein by reference in their entirety.

The cell culture supernatants are screened for the desired antibodies,preferentially by immunofluorescent staining of complement componentC5-expressing cells, by immunoblotting, by an enzyme immunoassay, e.g. asandwich assay or a dot-assay, or a radioimmunoassay.

For isolation of the antibodies, the immunoglobulins in the culturesupernatants or in the ascitic fluid may be concentrated, e.g., byprecipitation with ammonium sulfate, dialysis against hygroscopicmaterial such as polyethylene glycol, filtration through selectivemembranes, or the like. If necessary and/or desired, the antibodies arepurified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose and/or (immuno-) affinity chromatography, e.g. affinitychromatography with one or more surface polypeptides derived from acomplement component C5-expressing cell line, or with Protein-A or -G.

Another embodiment provides a process for the preparation of a bacterialcell line secreting antibodies directed against a C5 protein in asuitable mammal. For example a rabbit is immunized with pooled samplesfrom C5-expressing tissue or cells or C5 polypeptide or fragmentsthereof. A phage display library produced from the immunized rabbit isconstructed and panned for the desired antibodies in accordance withmethods well known in the art (such as, e.g., the methods disclosed inthe various references incorporated herein by reference).

Hybridoma cells secreting the monoclonal antibodies are also disclosed.The preferred hybridoma cells are genetically stable, secrete monoclonalantibodies described herein of the desired specificity, and can beexpanded from deep-frozen cultures by thawing and propagation in vitroor as ascites in vivo.

In another embodiment, a process is provided for the preparation of ahybridoma cell line secreting monoclonal antibodies against a complementcomponent C5 protein. In that process, a suitable mammal, for example aBalb/c mouse, is immunized with one or more polypeptides or antigenicfragments of C5 or with one or more polypeptides or antigenic fragmentsderived from a C5-expressing cell, the C5-expressing cell itself, or anantigenic carrier containing a purified polypeptide as described.Antibody-producing cells of the immunized mammal are grown briefly inculture or fused with cells of a suitable myeloma cell line. The hybridcells obtained in the fusion are cloned, and cell clones secreting thedesired antibodies are selected. For example, spleen cells of Balb/cmice immunized with a C5-expressing Chronic Lymphocytic Leukemia (CLL)cell line are fused with cells of the myeloma cell line PAI or themyeloma cell line Sp2/0-Ag 14. The obtained hybrid cells are thenscreened for secretion of the desired antibodies and positive hybridomacells are cloned.

Methods for preparing a hybridoma cell line include immunizing Balb/cmice by injecting subcutaneously and/or intraperitoneally an immunogeniccomposition containing human C5 protein (or an immunogenic fragmentthereof) several times, e.g., four to six times, over several months,e.g., between two and four months. Spleen cells from the immunized miceare taken two to four days after the last injection and fused with cellsof the myeloma cell line PAI in the presence of a fusion promoter,preferably polyethylene glycol. Preferably, the myeloma cells are fusedwith a three- to twenty-fold excess of spleen cells from the immunizedmice in a solution containing about 30% to about 50% polyethylene glycolof a molecular weight around 4000. After the fusion, the cells areexpanded in suitable culture media as described supra, supplemented witha selection medium, for example HAT medium, at regular intervals inorder to prevent normal myeloma cells from overgrowing the desiredhybridoma cells.

The antibodies and fragments thereof can be “chimeric.” Chimericantibodies and antigen-binding fragments thereof comprise portions fromtwo or more different species (e.g., mouse and human). Chimericantibodies can be produced with mouse variable regions of desiredspecificity spliced onto human constant domain gene segments (forexample, U.S. Pat. No. 4,816,567). In this manner, non-human antibodiescan be modified to make them more suitable for human clinicalapplication (e.g., methods for treating or preventing a complementassociated disorder in a human subject).

The monoclonal antibodies of the present disclosure include “humanized”forms of the non-human (e.g., mouse) antibodies. Humanized orCDR-grafted mAbs are particularly useful as therapeutic agents forhumans because they are not cleared from the circulation as rapidly asmouse antibodies and do not typically provoke an adverse immunereaction. Methods of preparing humanized antibodies are generally wellknown in the art. For example, humanization can be essentially performedfollowing the method of Winter and co-workers (see, e.g., Jones et al.(1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327;and Verhoeyen et al. (1988) Science 239:1534-1536), by substitutingrodent CDRs or CDR sequences for the corresponding sequences of a humanantibody. Also see, e.g., Staelens et al. (2006) Mol Immunol43:1243-1257. In some embodiments, humanized forms of non-human (e.g.,mouse) antibodies are human antibodies (recipient antibody) in whichhypervariable (CDR) region residues of the recipient antibody arereplaced by hypervariable region residues from a non-human species(donor antibody) such as a mouse, rat, rabbit, or non-human primatehaving the desired specificity, affinity, and binding capacity. In someinstances, framework region residues of the human immunoglobulin arealso replaced by corresponding non-human residues (so called “backmutations”). In addition, phage display libraries can be used to varyamino acids at chosen positions within the antibody sequence. Theproperties of a humanized antibody are also affected by the choice ofthe human framework. Furthermore, humanized and chimerized antibodiescan be modified to comprise residues that are not found in the recipientantibody or in the donor antibody in order to further improve antibodyproperties, such as, for example, affinity or effector function.

Fully human antibodies are also provided in the disclosure. The term“human antibody” includes antibodies having variable and constantregions (if present) derived from human germline immunoglobulinsequences. Human antibodies can include amino acid residues not encodedby human germline immunoglobulin sequences (e.g., mutations introducedby random or site-specific mutagenesis in vitro or by somatic mutationin vivo). However, the term “human antibody” does not include antibodiesin which CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences (i.e., humanized antibodies). Fully human or human antibodiesmay be derived from transgenic mice carrying human antibody genes(carrying the variable (V), diversity (D), joining (J), and constant (C)exons) or from human cells. For example, it is now possible to producetransgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production. (See, e.g., Jakobovits et al.(1993) Proc Natl Acad Sci USA 90:2551; Jakobovits et al. (1993) Nature362:255-258; Bruggemann et al. (1993) Year in Immunol. 7:33; andDuchosal et al. (1992) Nature 355:258.) Transgenic mice strains can beengineered to contain gene sequences from unrearranged humanimmunoglobulin genes. The human sequences may code for both the heavyand light chains of human antibodies and would function correctly in themice, undergoing rearrangement to provide a wide antibody repertoiresimilar to that in humans. The transgenic mice can be immunized with thetarget protein (e.g., a complement component C5 protein, fragmentsthereof, or cells expressing C5 protein) to create a diverse array ofspecific antibodies and their encoding RNA. Nucleic acids encoding theantibody chain components of such antibodies may then be cloned from theanimal into a display vector. Typically, separate populations of nucleicacids encoding heavy and light chain sequences are cloned, and theseparate populations then recombined on insertion into the vector, suchthat any given copy of the vector receives a random combination of aheavy and a light chain. The vector is designed to express antibodychains so that they can be assembled and displayed on the outer surfaceof a display package containing the vector. For example, antibody chainscan be expressed as fusion proteins with a phage coat protein from theouter surface of the phage. Thereafter, display packages can be screenedfor display of antibodies binding to a target.

In addition, human antibodies can be derived from phage-displaylibraries (Hoogenboom et al. (1991) J Mol Biol 227:381; Marks et al.(1991) J Mol Biol 222:581-597; and Vaughan et al. (1996) Nature Biotech14:309 (1996)). Synthetic phage libraries can be created which userandomized combinations of synthetic human antibody V-regions. Byselection on antigen fully human antibodies can be made in which theV-regions are very human-like in nature. See, e.g., U.S. Pat. Nos.6,794,132; 6,680,209; and 4,634,666, and Ostberg et al. (1983) Hybridoma2:361-367, the contents of each of which are incorporated herein byreference in their entirety.

For the generation of human antibodies, also see Mendez et al. (1998)Nature Genetics 15:146-156 and Green and Jakobovits (1998) J Exp Med188:483-495, the disclosures of which are hereby incorporated byreference in their entirety. Human antibodies are further discussed anddelineated in U.S. Pat. Nos. 5,939,598; 6,673,986; 6,114,598; 6,075,181;6,162,963; 6,150,584; 6,713,610; and 6,657,103 as well as U.S. PatentApplication Publication Nos. 20030229905 A1, 20040010810 A1, 20040093622A1, 20060040363 A1, 20050054055 A1, 20050076395 A1, and 20050287630 A1.See also International Patent Application Publication Nos. WO 94/02602,WO 96/34096, and WO 98/24893, and European Patent No. EP 0 463 151 B1.The disclosures of each of the above-cited patents, applications, andreferences are hereby incorporated by reference in their entirety.

In an alternative approach, others, including GenPharm International,Inc., have utilized a “minilocus” approach. In the minilocus approach,an exogenous Ig locus is mimicked through the inclusion of pieces(individual genes) from the Ig locus. Thus, one or more V_(H) genes, oneor more D_(H) genes, one or more J_(H) genes, a mu constant region, anda second constant region (preferably a gamma constant region) are formedinto a construct for insertion into an animal. This approach isdescribed in, e.g., U.S. Pat. Nos. 5,545,807; 5,545,806; 5,625,825;5,625,126; 5,633,425; 5,661,016; 5,770,429; 5,789,650; 5,814,318;5,591,669; 5,612,205; 5,721,367; 5,789,215; 5,643,763; 5,569,825;5,877,397; 6,300,129; 5,874,299; 6,255,458; and 7,041,871, thedisclosures of which are hereby incorporated by reference. See alsoEuropean Patent No. 0 546 073 B1, International Patent ApplicationPublication Nos. WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO98/24884, the disclosures of each of which are hereby incorporated byreference in their entirety. See further Taylor et al. (1992) NucleicAcids Res 20: 6287; Chen et al. (1993) Int Immunol 5:647; Tuaillon etal. (1993) Proc Natl Acad Sci USA 90: 3720-4; Choi et al. (1993) NatureGenetics 4: 117; Lonberg et al. (1994) Nature 368: 856-859; Taylor etal. (1994) Int Immunol 6: 579-591; Tuaillon et al. (1995) J Immunol 154:6453-65; Fishwild et al. (1996) Nature Biotechnol 14: 845; and Tuaillonet al. (2000) Eur J Immunol 10: 2998-3005, the disclosures of each ofwhich are hereby incorporated by reference in their entirety.

In certain embodiments, de-immunized anti-C5 antibodies orantigen-binding fragments thereof are provided. De-immunized antibodiesor antigen-binding fragments thereof are antibodies that have beenmodified so as to render the antibody or antigen-binding fragmentthereof non-immunogenic, or less immunogenic, to a given species (e.g.,to a human). De-immunization can be achieved by modifying the antibodyor antigen-binding fragment thereof utilizing any of a variety oftechniques known to those skilled in the art (see, e.g., PCT PublicationNos. WO 04/108158 and WO 00/34317). For example, an antibody orantigen-binding fragment thereof may be de-immunized by identifyingpotential T cell epitopes and/or B cell epitopes within the amino acidsequence of the antibody or antigen-binding fragment thereof andremoving one or more of the potential T cell epitopes and/or B cellepitopes from the antibody or antigen-binding fragment thereof, forexample, using recombinant techniques. The modified antibody orantigen-binding fragment thereof may then optionally be produced andtested to identify antibodies or antigen-binding fragments thereof thathave retained one or more desired biological activities, such as, forexample, binding affinity, but have reduced immunogenicity. Methods foridentifying potential T cell epitopes and/or B cell epitopes may becarried out using techniques known in the art, such as, for example,computational methods (see e.g., PCT Publication No. WO 02/069232), invitro or in silico techniques, and biological assays or physical methods(such as, for example, determination of the binding of peptides to MHCmolecules, determination of the binding of peptide:MHC complexes to theT cell receptors from the species to receive the antibody orantigen-binding fragment thereof, testing of the protein or peptideparts thereof using transgenic animals with the MHC molecules of thespecies to receive the antibody or antigen-binding fragment thereof, ortesting with transgenic animals reconstituted with immune system cellsfrom the species to receive the antibody or antigen-binding fragmentthereof, etc.). In various embodiments, the de-immunized anti-C5antibodies described herein include de-immunized antigen-bindingfragments, Fab, Fv, scFv, Fab′ and F(ab′)₂, monoclonal antibodies,murine antibodies, engineered antibodies (such as, for example,chimeric, single chain, CDR-grafted, humanized, and artificiallyselected antibodies), synthetic antibodies and semi-syntheticantibodies.

In some embodiments, a recombinant DNA comprising an insert coding for aheavy chain variable domain and/or for a light chain variable domain ofan anti-C5 antibody or a C5 protein-expressing cell line is produced.The term DNA includes coding single stranded DNAs, double stranded DNAsconsisting of said coding DNAs and of complementary DNAs thereto, orthese complementary (single stranded) DNAs themselves.

Furthermore, a DNA encoding a heavy chain variable domain and/or a lightchain variable domain of anti-C5 antibodies can be enzymatically orchemically synthesized to contain the authentic DNA sequence coding fora heavy chain variable domain and/or for the light chain variabledomain, or a mutant thereof. A mutant of the authentic DNA is a DNAencoding a heavy chain variable domain and/or a light chain variabledomain of the above-mentioned antibodies in which one or more aminoacids are deleted, inserted, or exchanged with one or more other aminoacids. Preferably said modification(s) are outside the CDRs of the heavychain variable domain and/or the CDRs of the light chain variable domainof the antibody in humanization and expression optimizationapplications. The term mutant DNA also embraces silent mutants whereinone or more nucleotides are replaced by other nucleotides with the newcodons coding for the same amino acid(s). The term mutant sequence alsoincludes a degenerate sequence. Degenerate sequences are degeneratewithin the meaning of the genetic code in that an unlimited number ofnucleotides are replaced by other nucleotides without resulting in achange of the amino acid sequence originally encoded. Such degeneratesequences may be useful due to their different restriction sites and/orfrequency of particular codons which are preferred by the specific host,particularly E. coli, to obtain an optimal expression of the heavy chainmurine variable domain and/or a light chain murine variable domain.

The term mutant is intended to include a DNA mutant obtained by in vitromutagenesis of the authentic DNA according to methods known in the art.For the assembly of complete tetrameric immunoglobulin molecules and theexpression of chimeric antibodies, the recombinant DNA inserts codingfor heavy and light chain variable domains are fused with thecorresponding DNAs coding for heavy and light chain constant domains,then transferred into appropriate host cells, for example afterincorporation into hybrid vectors.

Recombinant DNAs including an insert coding for a heavy chain murinevariable domain of an anti-C5 antibody-expressing cell line fused to ahuman constant domain IgG, for example γ1, γ2, γ3 or γ4, in particularembodiments γ1 or γ4, may be used. Recombinant DNAs including an insertcoding for a light chain murine variable domain of an antibody fused toa human constant domain κ or λ, preferably κ, are also provided.

Another embodiment pertains to recombinant DNAs coding for a recombinantpolypeptide wherein the heavy chain variable domain and the light chainvariable domain are linked by way of a spacer group, optionallycomprising a signal sequence facilitating the processing of the antibodyin the host cell and/or a DNA sequence encoding a peptide facilitatingthe purification of the antibody and/or a cleavage site and/or a peptidespacer and/or an agent.

Accordingly, the monoclonal antibodies or antigen-binding fragments ofthe disclosure can be naked antibodies or antigen-binding fragments thatare not conjugated to other agents, for example, a therapeutic agent ordetectable label. Alternatively, the monoclonal antibody orantigen-binding fragment can be conjugated to an agent such as, forexample, a cytotoxic agent, a small molecule, a hormone, an enzyme, agrowth factor, a cytokine, a ribozyme, a peptidomimetic, a chemical, aprodrug, a nucleic acid molecule including coding sequences (such asantisense, RNAi, gene-targeting constructs, etc.), or a detectable label(e.g., an NMR or X-ray contrasting agent, fluorescent molecule, etc.).In certain embodiments, an anti-C5 antibody or antigen-binding fragment(e.g., Fab, Fv, single-chain (scFv), Fab′, and F(ab′)₂) is linked to amolecule that increases the half-life of the antibody or antigen-bindingfragment (see above).

Several possible vector systems are available for the expression ofcloned heavy chain and light chain genes in mammalian cells. One classof vectors relies upon the integration of the desired gene sequencesinto the host cell genome. Cells which have stably integrated DNA can beselected by simultaneously introducing drug resistance genes such as E.coli gpt (Mulligan and Berg (1981) Proc Natl Acad Sci USA, 78:2072-2076)or Tn5 neo (Southern and Berg (1982) J Mol Appl Genet 1:327-341). Theselectable marker gene can be either linked to the DNA gene sequences tobe expressed, or introduced into the same cell by co-transfection(Wigler et al. (1979) Cell 16:777-785). A second class of vectorsutilizes DNA elements which confer autonomously replicating capabilitiesto an extrachromosomal plasmid. These vectors can be derived from animalviruses, such as bovine papillomavirus (Sarver et al. (1982) Proc NatlAcad Sci USA, 79:7147-7151), polyoma virus (Deans et al. (1984) ProcNatl Acad Sci USA 81:1292-1296), or SV40 virus (Lusky and Botchan (1981)Nature 293:79-81).

Since an immunoglobulin cDNA is comprised only of sequences representingthe mature mRNA encoding an antibody protein, additional gene expressionelements regulating transcription of the gene and processing of the RNAare required for the synthesis of immunoglobulin mRNA. These elementsmay include splice signals, transcription promoters, including induciblepromoters, enhancers, and termination signals. cDNA expression vectorsincorporating such elements include those described by Okayama and Berg(1983) Mol Cell Biol 3:280-289; Cepko et al. (1984) Cell 37:1053-1062;and Kaufman (1985) Proc Natl Acad Sci USA 82:689-693.

As is evident from the disclosure, the anti-C5 antibodies can be used intherapies (e.g., therapies for a complement associated disorder),including combination therapies.

In the therapeutic embodiments of the present disclosure, bispecificantibodies are contemplated. Bispecific antibodies are monoclonal,preferably human or humanized, antibodies that have bindingspecificities for at least two different antigens. In the present case,one of the binding specificities is for the human complement componentC5 antigen and the other one is for any other antigen.

Methods for making bispecific antibodies are within the purview of thoseskilled in the art. Traditionally, the recombinant production ofbispecific antibodies is based on the co-expression of twoimmunoglobulin heavy-chain/light-chain pairs, where the two heavy chainshave different specificities (Milstein and Cuello (1983) Nature305:537-539). Antibody variable domains with the desired bindingspecificities (antibody-antigen combining sites) can be fused toimmunoglobulin constant domain sequences. The fusion preferably is withan immunoglobulin heavy-chain constant domain, including at least partof the hinge, C_(H)2, and C_(H)3 regions. DNAs encoding theimmunoglobulin heavy-chain fusions and, if desired, the immunoglobulinlight chain, are inserted into separate expression vectors, and areco-transfected into a suitable host organism. For further details ofillustrative currently known methods for generating bispecificantibodies see, e.g., Suresh et al. (1986) Methods Enzymol 121:210-228;PCT Publication No. WO 96/27011; Brennan et al. (1985) Science229:81-83; Shalaby et al. J Exp Med (1992) 175:217-225; Kostelny et al.(1992) J Immunol 148(5):1547-1553; Hollinger et al. (1993) Proc NatlAcad Sci USA 90:6444-6448; Gruber et al. (1994) J Immunol 152:5368-5474;and Tutt et al. (1991) J Immunol 147:60-69. Bispecific antibodies alsoinclude cross-linked or heteroconjugate antibodies. Heteroconjugateantibodies may be made using any convenient cross-linking methods.Suitable cross-linking agents are well known in the art, and aredisclosed in U.S. Pat. No. 4,676,980, along with a number ofcross-linking techniques.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. See, e.g., Kostelny et al. (1992) J Immunol148(5):1547-1553. The leucine zipper peptides from the Fos and Junproteins may be linked to the Fab′ portions of two different antibodiesby gene fusion. The antibody homodimers may be reduced at the hingeregion to form monomers and then re-oxidized to form the antibodyheterodimers. This method can also be utilized for the production ofantibody homodimers. The “diabody” technology described by Hollinger etal. (1993) Proc Natl Acad Sci USA 90:6444-6448 has provided analternative mechanism for making bispecific antibody fragments. Thefragments comprise a heavy-chain variable domain (VH) connected to alight-chain variable domain (VL) by a linker which is too short to allowpairing between the two domains on the same chain. Accordingly, the VHand VL domains of one fragment are forced to pair with the complementaryVL and VH domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (scFv) dimers has also beenreported. See, e.g., Gruber et al. (1994) J Immunol 152:5368-5374.Alternatively, the antibodies can be “linear antibodies” as describedin, e.g., Zapata et al. (1995) Protein Eng 8(10):1057-1062. Briefly,these antibodies comprise a pair of tandem Fd segments(V_(H)-C_(H)1-V_(H)-C_(H)1) which form a pair of antigen bindingregions. Linear antibodies can be bispecific or monospecific.

The disclosure also embraces variant forms of bispecific antibodies suchas the tetravalent dual variable domain immunoglobulin (DVD-Ig)molecules described in Wu et al. (2007) Nat Biotechnol 25(11):1290-1297.The DVD-Ig molecules are designed such that two different light chainvariable domains (VL) from two different parent antibodies are linked intandem directly or via a short linker by recombinant DNA techniques,followed by the light chain constant domain. Methods for generatingDVD-Ig molecules from two parent antibodies are further described in,e.g., PCT Publication Nos. WO 08/024,188 and WO 07/024,715, thedisclosures of each of which are incorporated herein by reference intheir entirety.

Methods for Treatment

The above-described compositions (e.g., any of the high concentrationantibody solutions) are useful in, inter alia, methods for treating orpreventing a variety of complement-associated disorders (e.g.,AP-associated disorders or CP-associated disorders) in a subject. Thecompositions can be administered to a subject, e.g., a human subject,using a variety of methods that depend, in part, on the route ofadministration. The route can be, e.g., intravenous injection orinfusion (IV), subcutaneous injection (SC), intraperitoneal (IP)injection, intraocular injection, intraarticular injection, orintramuscular injection (IM).

In some embodiments, a high concentration antibody solution describedherein is therapeutically delivered to a subject by way of localadministration. As used herein, “local administration” or “localdelivery,” refers to delivery that does not rely upon transport of thecomposition or active agent (e.g., an anti-C5 antibody) to its intendedtarget tissue or site via the vascular system. For example, thecomposition may be delivered by injection or implantation of thecomposition or agent or by injection or implantation of a devicecontaining the composition or agent. Following local administration inthe vicinity of a target tissue or site, the composition or agent, orone or more components thereof, may diffuse to the intended targettissue or site.

In some embodiments, a high concentration antibody solution can belocally administered to a joint (e.g., an articulated joint). Forexample, in embodiments where the complement-associated disorder isarthritis, the solution can be administered directly to a joint (e.g.,into a joint space) or in the vicinity of a joint. Examples ofintraarticular joints to which a high concentration antibody solutiondescribed herein can be locally administered include, e.g., the hip,knee, elbow, wrist, sternoclavicular, temperomandibular, carpal, tarsal,ankle, and any other joint subject to arthritic conditions. A highconcentration solution described herein can also be administered tobursa such as, e.g., acromial, bicipitoradial, cubitoradial, deltoid,infrapatellar, ischial, and any other bursa known in the art ofmedicine.

In some embodiments, a high concentration antibody solution describedherein can be locally administered to the eye, e.g., to treat patientsafflicted with a complement-associated disorder of the eye such as wetor dry AMD. As used herein, the term “eye” refers to any and allanatomical tissues and structures associated with an eye. The eye has awall composed of three distinct layers: the outer sclera, the middlechoroid layer, and the inner retina. The chamber behind the lens isfilled with a gelatinous fluid referred to as the vitreous humor. At theback of the eye is the retina, which detects light. The cornea is anoptically transparent tissue, which conveys images to the back of theeye. The cornea includes one pathway for the permeation of drugs intothe eye. Other anatomical tissue structures associated with the eyeinclude the lacrimal drainage system, which includes a secretory system,a distributive system and an excretory system. The secretory systemcomprises secretors that are stimulated by blinking and temperaturechange due to tear evaporation and reflex secretors that have anefferent parasympathetic nerve supply and secrete tears in response tophysical or emotional stimulation. The distributive system includes theeyelids and the tear meniscus around the lid edges of an open eye, whichspread tears over the ocular surface by blinking, thus reducing dryareas from developing.

In some embodiments, a high concentration antibody solution describedherein is administered to the posterior chamber of the eye. In someembodiments, a high concentration antibody solution is administeredintravitreally. In some embodiments, a high concentration antibodysolution described herein is administered transsclerally.

It is understood that in some embodiments a high concentration antibodysolution described herein can be administered systemically for use intreating, e.g., RA, wet or dry AMD, or any other complement-associateddisorder described herein.

A suitable dose of a high concentration antibody solution describedherein, which dose is capable of treating or preventing acomplement-associated disorder in a subject, can depend on a variety offactors including, e.g., the age, sex, and weight of a subject to betreated and the particular inhibitor compound used. For example, adifferent dose of an anti-C5 antibody (and thus a differentconcentration of the antibody in solution or a different volume of ahigh concentration antibody solution) may be required to treat anelderly subject with RA as compared to the dose of an anti-C5 antibodythat is required to treat a younger subject. Other factors affecting thedose administered to the subject include, e.g., the type or severity ofthe complement-associated disorder. For example, a subject having RA mayrequire administration of a different dosage of a high concentrationantibody solution described herein than a subject with AMD. Otherfactors can include, e.g., other medical disorders concurrently orpreviously affecting the subject, the general health of the subject, thegenetic disposition of the subject, diet, time of administration, rateof excretion, drug combination, and any other additional therapeuticsthat are administered to the subject. It should also be understood thata specific dosage and treatment regimen for any particular subject isgenerally governed by the judgment of the treating medical practitioner(e.g., doctor or nurse).

An anti-C5 antibody as part of a high concentration antibody solutiondescribed herein can be administered as a fixed dose, or in a milligramper kilogram (mg/kg) dose. In some embodiments where localadministration is preferred, a dose can be selected that results inlocal inhibition of C5 cleavage (through the action of the anti-C5antibody), but with no substantial effect on systemic complementactivity. In some embodiments, the dose can also be chosen to reduce oravoid production of antibodies or other host immune responses againstthe therapeutic antibodies in the composition. While in no way intendedto be limiting, exemplary dosages of an anti-C5 antibody locallyadministered to the eye of a patient afflicted with AMD include 0.5 mgto 5 mg (e.g., at least 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4,4.5, 4.6, 4.7, 4.8, 4.9. or 5 mg) per dose. The dose, or pharmaceuticalunit dosage form, can be provided to the eye of the patient in a volumeof, e.g., up to 50 (e.g., one, two, three, four, five, six, seven,eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or 50) microliters. Accordingly, thedisclosure embraces and features pharmaceutical unit dosage forms of ananti-C5 antibody for use in treating AMD (e.g., wet or dry AMD) in apatient by intravitreal injection, which dosage form includes between0.5 mg to 5 mg, inclusive in a volume of not more than 50 microliters.

While in no way intended to be limiting, exemplary dosages of an anti-C5antibody locally administered to a joint (e.g., an articulated joint) ofa patient afflicted with rheumatoid arthritis (RA) include 0.5 to 10 mg(e.g., at least 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3,3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5,4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6,6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5,7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9,9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10 mg) per dose. Thedose, or pharmaceutical unit dosage form, can be provided to a joint ofthe patient in a volume of, e.g., up to 500 (e.g., one, two, three,four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330,340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470,480, 490, or 500) microliters. Accordingly, the disclosure embraces andfeatures pharmaceutical unit dosage forms of an anti-C5 antibody for usein treating RA in a patient by intraarticular injection, which dosageform includes between 0.5 mg to 10 mg, inclusive in a volume of not morethan 500 microliters.

Methods for detecting systemic hemolytic activity, as well as inhibitionof said activity, are well known in the art and are described herein.

In some embodiments, the concentrated solution of an anti-C5 antibodycan be diluted into a pharmaceutically-acceptable diluent for, e.g.,systemic delivery of the antibody to the subject. While in no wayintended to be limiting, exemplary dosages of an anti-C5 antibody to beadministered systemically to treat complement-associated disorderinclude, e.g., 1-100 μg/kg, 0.5-50 μg/kg, 0.1-100 μg/kg, 0.5-25 μg/kg,1-20 μg/kg, and 1-10 μg/kg, 1-100 mg/kg, 0.5-50 mg/kg, 0.1-100 mg/kg,0.5-25 mg/kg, 1-20 mg/kg, and 1-10 mg/kg. Exemplary dosages of anantibody described herein include, without limitation, 0.1 μg/kg, 0.5μg/kg, 1.0 μg/kg, 2.0 μg/kg, 4 μg/kg, and 8 μg/kg, 0.1 mg/kg, 0.5 mg/kg,1.0 mg/kg, 2.0 mg/kg, 4 mg/kg, and 8 mg/kg.

A therapeutically-effective amount of an anti-C5 antibody describedherein can be readily determined by one of ordinary skill in the artbased, in part, on the effect of the administered antibody, or thecombinatorial effect of the antibody and one or more additional activeagents, if more than one agent is used. A therapeutically effectiveamount of an antibody described herein can also vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the antibody (and one or more additionalactive agents) to elicit a desired response in the individual, e.g.,amelioration of at least one condition parameter, e.g., amelioration ofat least one symptom of the complement-associated disorder. For example,a therapeutically effective amount of a C5-binding polypeptide caninhibit (lessen the severity of or eliminate the occurrence of) and/orprevent a particular disorder, and/or any one of the symptoms of theparticular disorder known in the art or described herein. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the composition are outweighed by thetherapeutically beneficial effects.

Suitable human doses of any of the anti-C5 antibodies described hereincan further be evaluated in, e.g., Phase I dose escalation studies. See,e.g., van Gurp et al. (2008) Am J Transplantation 8(8):1711-1718;Hanouska et al. (2007) Clin Cancer Res 13(2, part 1):523-531; andHetherington et al. (2006) Antimicrobial Agents and Chemotherapy 50(10):3499-3500.

The terms “therapeutically effective amount” or “therapeuticallyeffective dose,” or similar terms used herein are intended to mean anamount of an agent (e.g., an anti-C5 antibody) that will elicit thedesired biological or medical response (e.g., an improvement in one ormore symptoms of a complement-associated disorder). In some embodiments,a high concentration antibody solution described herein contains atherapeutically effective amount of the anti-C5 antibody. In someembodiments, the high concentration antibody solution described hereincontains an anti-C5 antibody and one or more (e.g., one, two, three,four, five, six, seven, eight, nine, 10, or 11 or more) additionaltherapeutic agents such that the composition as a whole istherapeutically effective. For example, a high concentration antibodysolution can contain anti-C5 antibody and a VEGF inhibitor (e.g., ananti-VEGF antibody such as bevacizumab), wherein the anti-C5 antibodyand VEGF inhibitor are each at a concentration that when combined aretherapeutically effective for treating or preventing acomplement-associated disorder in a subject.

Toxicity and therapeutic efficacy of such compositions can be determinedby known pharmaceutical procedures in cell cultures or experimentalanimals (e.g., animal models of any of the complement-associateddisorders described herein). These procedures can be used, e.g., fordetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD₅₀/ED₅₀. An anti-C5antibody that exhibits a high therapeutic index is preferred. Whilecompositions that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue and to minimize potential damage to normal cellsand, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch antibodies lies generally within a range of circulatingconcentrations of the anti-C5 antibody that include the ED₅₀ with littleor no toxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For ananti-C5 antibody used as described herein (e.g., for treating orpreventing a complement-associated disorder), the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose can be formulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography or byELISA.

In some embodiments, the methods can be performed in conjunction withother therapies for complement-associated disorders. For example, thecomposition can be administered to a subject at the same time, prior to,or after, plasmapheresis, IVIG therapy, plasma replacement, or plasmaexchange. See, e.g., Appel et al. (2005) J Am Soc Nephrol 16:1392-1404.In some embodiments, a high concentration antibody solution describedherein is not administered in conjunction with IVIG. In someembodiments, e.g., for patients with aHUS, the composition can beadministered to a subject at the same time, prior to, or after, a kidneytransplant.

A “subject,” as used herein, can be any mammal. For example, a subjectcan be a human, a non-human primate (e.g., monkey, baboon, orchimpanzee), a horse, a cow, a pig, a sheep, a goat, a dog, a cat, arabbit, a guinea pig, a gerbil, a hamster, a rat, or a mouse. In someembodiments, the subject is an infant (e.g., a human infant).

As used herein, a subject “in need of prevention,” “in need oftreatment,” or “in need thereof,” refers to one, who by the judgment ofan appropriate medical practitioner (e.g., a doctor, a nurse, or a nursepractitioner in the case of humans; a veterinarian in the case ofnon-human mammals), would reasonably benefit from a given treatment(such as treatment with a solution comprising a high concentration of ananti-C5 antibody).

As described above, the high concentration antibody solutions describedherein can be used to treat a variety of complement-associated disorderssuch as, e.g., AP-associated disorders and/or CP-associated disorders.Such disorders include, without limitation, rheumatoid arthritis (RA);antiphospholipid antibody syndrome; lupus nephritis;ischemia-reperfusion injury; atypical hemolytic uremic syndrome (aHUS);typical or infectious hemolytic uremic syndrome (tHUS); dense depositdisease (DDD); paroxysmal nocturnal hemoglobinuria (PNH); neuromyelitisoptica (NMO); multifocal motor neuropathy (MMN); multiple sclerosis(MS); macular degeneration (e.g., age-related macular degeneration(AMD)); hemolysis, elevated liver enzymes, and low platelets (HELLP)syndrome; thrombotic thrombocytopenic purpura (TTP); spontaneous fetalloss; Pauci-immune vasculitis; epidermolysis bullosa; recurrent fetalloss; and traumatic brain injury. (See, e.g., Holers (2008)Immunological Reviews 223:300-316 and Holers and Thurman (2004)Molecular Immunology 41:147-152.) In some embodiments, thecomplement-associated disorder is a complement-associated vasculardisorder such as, but not limited to, a diabetes-associated vasculardisorder (e.g., of the eye), central retinal vein occlusion, acardiovascular disorder, myocarditis, a cerebrovascular disorder, aperipheral (e.g., musculoskeletal) vascular disorder, a renovasculardisorder, a mesenteric/enteric vascular disorder, revascularization totransplants and/or replants, vasculitis, Henoch-Schönlein purpuranephritis, systemic lupus erythematosus-associated vasculitis,vasculitis associated with rheumatoid arthritis, immune complexvasculitis, Takayasu's disease, dilated cardiomyopathy, diabeticangiopathy, Kawasaki's disease (arteritis), venous gas embolus (VGE),and restenosis following stent placement, rotational atherectomy, andpercutaneous transluminal coronary angioplasty (PTCA). (See, e.g., U.S.patent application publication no. 20070172483.) Additionalcomplement-associated disorders include, without limitation, myastheniagravis, cold agglutinin disease, dermatomyositis, Graves' disease,atherosclerosis, Alzheimer's disease, Guillain-Barré Syndrome, Degos'disease, graft rejection (e.g., transplant rejection), sepsis, burn(e.g., severe burn), systemic inflammatory response sepsis, septicshock, spinal cord injury, glomerulonephritis, Hashimoto's thyroiditis,type I diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia(AIHA), idiopathic thrombocytopenic purpura (ITP), Goodpasture syndrome,antiphospholipid syndrome (APS), and catastrophic APS (CAPS). In someembodiments, the high concentration antibody solutions described hereincan be used in methods for treating thrombotic microangiopathy (TMA),e.g., TMA associated with a complement-associated disorder such as anyof the complement-associated disorders described herein.

Complement-associated disorders also include complement-associatedpulmonary disorders such as, but not limited to, asthma, bronchitis, achronic obstructive pulmonary disease (COPD), an interstitial lungdisease, α-1 anti-trypsin deficiency, emphysema, bronchiectasis,bronchiolitis obliterans, alveolitis, sarcoidosis, pulmonary fibrosis,and collagen vascular disorders.

As used herein, a subject “at risk for developing acomplement-associated disorder” (e.g., an AP-associated disorder or aCP-associated disorder) is a subject having one or more (e.g., two,three, four, five, six, seven, or eight or more) risk factors fordeveloping the disorder. Risk factors will vary depending on theparticular complement-associated disorder, but are well known in the artof medicine. For example, risk factors for developing DDD include, e.g.,a predisposition to develop the condition, i.e., a family history of thecondition or a genetic predisposition to develop the condition such as,e.g., one or more mutations in the gene encoding complement factor H(CFH), complement factor H-related 5 (CFHR5), and/or complementcomponent C3 (C3). Such DDD-associated mutations as well methods fordetermining whether a subject carries one or more of the mutations areknown in the art and described in, e.g., Licht et al. (2006) Kidney Int70:42-50; Zipfel et al. (2006) “The role of complement inmembranoproliferative glomerulonephritis,” In: Complement and KidneyDisease, Springer, Berlin, pages 199-221; Ault et al. (1997) J Biol Chem272:25168-75; Abrera-Abeleda et al. (2006) J Med Genet 43:582-589;Poznansky et al. (1989) J Immunol 143:1254-1258; Jansen et al. (1998)Kidney Int 53:331-349; and Hegasy et al. (2002) Am Pathol 161:2027-2034.Thus, a human at risk for developing DDD can be, e.g., one who has oneor more DDD-associated mutations in the gene encoding CFH or one with afamily history of developing the disease.

Risk factors for TTP are well known in the art of medicine and include,e.g., a predisposition to develop the condition, i.e., a family historyof the condition or a genetic predisposition to develop the conditionsuch as, e.g., one or more mutations in the ADAMTS13 gene. ADAMTS13mutations associated with TTP are reviewed in detail in, e.g., Levy etal. (2001) Nature 413:488-494; Kokame et al. (2004) Semin Hematol41:34-40; Licht et al. (2004) Kidney Int 66:955-958; and Noris et al.(2005) J Am Soc Nephrol 16:1177-1183. Risk factors for TTP also includethose conditions or agents that are known to precipitate TTP, or TTPrecurrence, such as, but not limited to, cancer, bacterial infections(e.g., Bartonella sp. infections), viral infections (e.g., HIV andKaposi's sarcoma virus), pregnancy, or surgery. See, e.g., Avery et al.(1998) Am J Hematol 58:148-149 and Tsai, supra. TTP, or recurrence ofTTP, has also been associated with the use of certain therapeutic agents(drugs) including, e.g., ticlopidine, FK506, corticosteroids, tamoxifen,or cyclosporin A (see, e.g., Gordon et al. (1997) Sem in Hematol34(2):140-147). Hereinafter, such manifestations of TTP may be, whereappropriate, referred to as, e.g., “infection-associated TTP,”“pregnancy-associated TTP,” or “drug-associated TTP.” Thus, a human atrisk for developing TTP can be, e.g., one who has one or moreTTP-associated mutations in the ADAMTS13 gene. A human at risk fordeveloping a recurrent form of TTP can be one, e.g., who has had TTP andhas an infection, is pregnant, or is undergoing surgery.

Risk factors for aHUS are well known in the art of medicine and include,e.g., a predisposition to develop the condition, i.e., a family historyof the condition or a genetic predisposition to develop the conditionsuch as, e.g., one or more mutations in complement Factor H(CFH),membrane cofactor protein (MCP; CD46), C4b-binding protein, complementfactor B (CFB), or complement factor I (CFI). (See, e.g., Warwicker etal. (1998) Kidney Int 53:836-844; Richards et al. (2001) Am J Hum Genet68:485-490; Caprioli et al. (2001) Am Soc Nephrol 12:297-307; Neuman etal. (2003) J Med Genet 40:676-681; Richards et al. (2006) Proc Natl AcadSci USA 100:12966-12971; Fremeaux-Bacchi et al. (2005) J Am Soc Nephrol17:2017-2025; Esparza-Gordillo et al. (2005) Hum Mol Genet 14:703-712;Goicoechea de Jorge et al. (2007) Proc Natl Acad Sci USA 104(1):240-245;Blom et al. (2008) J Immunol 180(9):6385-91; and Fremeaux-Bacchi et al.(2004) J Medical Genet 41:e84). (See also Kavanagh et al. (2006) supra.)Risk factors also include, e.g., infection with Streptococcuspneumoniae, pregnancy, cancer, exposure to anti-cancer agents (e.g.,quinine, mitomycin C, cisplatin, or bleomycin), exposure toimmunotherapeutic agents (e.g., cyclosporine, OKT3, or interferon),exposure to anti-platelet agents (e.g., ticlopidine or clopidogrel), HIVinfection, transplantation, autoimmune disease, and combinedmethylmalonic aciduria and homocystinuria (cblC). See, e.g.,Constantinescu et al. (2004) Am J Kidney Dis 43:976-982; George (2003)Curr Opin Hematol 10:339-344; Gottschall et al. (1994) Am Hematol47:283-289; Valavaara et al. (1985) Cancer 55:47-50; Miralbell et al.(1996) J Clin Oncol 14:579-585; Dragon-Durey et al. (2005) J Am SocNephrol 16:555-63; and Becker et al. (2004) Clin Infect Dis39:S267-S275.

Risk factors for HELLP are well known in the art of medicine andinclude, e.g., multiparous pregnancy, maternal age over 25 years,Caucasian race, the occurrence of preeclampsia or HELLP in a previouspregnancy, and a history of poor pregnancy outcome. (See, e.g., Sahin etal. (2001) Nagoya Med J 44(3):145-152; Sullivan et al. (1994) Am JObstet Gynecol 171:940-943; and Padden et al. (1999) Am Fam Physician60(3):829-836.) For example, a pregnant, Caucasian woman who developedpreeclampsia during a first pregnancy can be one at risk for developingHELLP syndrome during, or following, a second pregnancy.

Risk factors for CAD are well known in the art of medicine and include,e.g., conditions or agents that are known to precipitate CAD, or CADrecurrence, such as, but not limited to, neoplasms or infections (e.g.,bacterial and viral infections). Conditions known to be associated withthe development of CAD include, e.g., HIV infection (and AIDS),hepatitis C infection, Mycoplasma pneumonia infection, Epstein-Barrvirus (EBV) infection, cytomegalovirus (CMV) infection, rubella, orinfectious mononucleosis. Neoplasms associated with CAD include, withoutlimitation, non-Hodgkin's lymphoma. Hereinafter, such manifestations ofCAD may be, where appropriate, referred to as, e.g.,“infection-associated CAD” or “neoplasm-associated CAD.” Thus, a humanat risk for developing CAD can be, e.g., one who has an HIV infection,rubella, or a lymphoma. See also, e.g., Gertz (2006) Hematology 1:19-23;Horwitz et al. (1977) Blood 50:195-202; Finland and Barnes (1958) AMAArch Intern Med 191:462-466; Wang et al. (2004) Acta Paediatr Taiwan45:293-295; Michaux et al. (1998) Ann Hematol 76:201-204; and Chang etal. (2004) Cancer Genet Cytogenet 152:66-69.

Risk factors for myasthenia gravis (MG) are well known in the art ofmedicine and include, e.g., a predisposition to develop the condition,i.e., a family history of the condition or a genetic predisposition todevelop the condition such as familial MG. For example, some HLA typesare associated with an increased risk for developing MG. Risk factorsfor MG include the ingestion or exposure to certain MG-inducing drugssuch as, but not limited to, D-penicillamine. See, e.g., Drosos et al.(1993) Clin Exp Rheumatol 11(4):387-91 and Kaeser et al. (1984) ActaNeurol Scand Suppl 100:39-47. As MG can be episodic, a subject who haspreviously experienced one or more symptoms of having MG can be at riskfor relapse. Thus, a human at risk for developing MG can be, e.g., onewho has a family history of MG and/or one who has ingested or beenadministered an MG-inducing drug such as D-penicillamine.

As used herein, a subject “at risk for developing CAPS” is a subjecthaving one or more (e.g., two, three, four, five, six, seven, or eightor more) risk factors for developing the disorder. Approximately 60% ofthe incidences of CAPS are preceded by a precipitating event such as aninfection. Thus, risk factors for CAPS include those conditions known toprecipitate CAPS such as, but not limited to, certain cancers (e.g.,gastric cancer, ovarian cancer, lymphoma, leukemia, endometrial cancer,adenocarcinoma, and lung cancer), pregnancy, puerperium,transplantation, primary APS, rheumatoid arthritis (RA), systemic lupuserythematosus (SLE), surgery (e.g., eye surgery), and certaininfections. Infections include, e.g., parvovirus B19 infection andhepatitis C infection. Hereinafter, such manifestations of CAPS may bereferred to as, e.g., “cancer-associated CAPS,”“transplantation-associated CAPS,” “RA-associated CAPS,”“infection-associated CAPS,” or “SLE-associated CAPS.” See, e.g.,Soltész et al. (2000) Haematologia (Budep) 30(4):303-311; Ideguchi etal. (2007) Lupus 16(1):59-64; Manner et al. (2008) Am J Med Sci335(5):394-7; Miesbach et al. (2006) Autoimmune Rev 6(2):94-7;Gómez-Puerta et al. (2006) Autoimmune Rev 6(2):85-8; Gómez-Puerta et al.(2006) Semin Arthritis Rheum 35(5):322-32; Kasamon et al. (2005)Haematologia 90(3):50-53; Atherson et al. (1998) Medicine 77(3):195-207;and Canpolat et al. (2008) Clin Pediatr 47(6):593-7. Thus, a human atrisk for developing CAPS can be, e.g., one who has primary CAPS and/or acancer that is known to be associated with CAPS.

From the above it will be clear that subjects “at risk for developing acomplement-associated disorder” (e.g., an AP-associated disorder or aCP-associated disorder) are not all the subjects within a species ofinterest.

A subject “suspected of having a complement-associated disorder” (e.g.,an alternative complement pathway-associated disorder) is one having oneor more (e.g., two, three, four, five, six, seven, eight, nine, or 10 ormore) symptoms of the disease. Symptoms of these disorders will varydepending on the particular disorder, but are known to those of skill inthe art of medicine. For example, symptoms of DDD include, e.g.: one orboth of hematuria and proteinuria; acute nephritic syndrome; drusendevelopment and/or visual impairment; acquired partial lipodystrophy andcomplications thereof; and the presence of serum C3 nephritic factor(C3NeF), an autoantibody directed against C3bBb, the C3 convertase ofthe alternative complement pathway. (See, e.g., Appel et al. (2005),supra). Symptoms of aHUS include, e.g., severe hypertension,proteinuria, uremia, lethargy/fatigue, irritability, thrombocytopenia,microangiopathic hemolytic anemia, and renal function impairment (e.g.,acute renal failure). Symptoms of TTP include, e.g., microthrombi,thrombocytopenia, fever, low ADAMTS13 metalloproteinase expression oractivity, fluctuating central nervous system abnormalities, renalfailure, microangiopathic hemolytic anemia, bruising, purpura, nauseaand vomiting (e.g., resulting from ischemia in the GI tract or fromcentral nervous system involvement), chest pain due to cardiac ischemia,seizures, and muscle and joint pain. Symptoms of RA can include, e.g.,stiffness, swelling, fatigue, anemia, weight loss, fever, and often,crippling pain. Some common symptoms of rheumatoid arthritis includejoint stiffness upon awakening that lasts an hour or longer; swelling ina specific finger or wrist joints; swelling in the soft tissue aroundthe joints; and swelling on both sides of the joint. Swelling can occurwith or without pain, and can worsen progressively or remain the samefor years before progressing. Symptoms of HELLP are known in the art ofmedicine and include, e.g., malaise, epigastric pain, nausea, vomiting,headache, right upper quadrant pain, hypertension, proteinuria, blurredvision, gastrointestinal bleeding, hypoglycemia, paresthesia, elevatedliver enzymes/liver damage, anemia (hemolytic anemia), and low plateletcount, any of which in combination with pregnancy or recent pregnancy.(See, e.g., Tomsen (1995) Am J Obstet Gynecol 172:1876-1890; Sibai(1986) Am J Obstet Gynecol 162:311-316; and Padden (1999), supra.)Symptoms of PNH include, e.g., hemolytic anemia (a decreased number ofred blood cells), hemoglobinuria (the presence of hemoglobin in theurine particularly evident after sleeping), and hemoglobinemia (thepresence of hemoglobin in the bloodstream). PNH-afflicted subjects areknown to have paroxysms, which are defined here as incidences ofdark-colored urine, dysphagia, fatigue, erectile dysfunction,thrombosis, and recurrent abdominal pain.

Symptoms of CAPS are well known in the art of medicine and include,e.g., histopathological evidence of multiple small vessel occlusions;the presence of antiphospholipid antibodies (usually at high titer),vascular thromboses, severe multi-organ dysfunction, malignanthypertension, acute respiratory distress syndrome, disseminatedintravascular coagulation, microangiopathic hemolytic anemia,schistocytes, and thrombocytopenia. CAPS can be distinguished from APSin that patients with CAPS generally present with severe multiple organdysfunction or failure, which is characterized by rapid, diffuse smallvessel ischemia and thromboses predominantly affecting the parenchymalorgans. In contrast, APS is associated with single venous or arterialmedium-to-large blood vessel occlusions. Symptoms of MG include, e.g.,fatigability and a range of muscle weakness-related conditionsincluding: ptosis (of one or both eyes), diplopia, unstable gait,depressed or distorted facial expressions, and difficulty chewing,talking, or swallowing. In some instances, a subject can present withpartial or complete paralysis of the respiratory muscles. Symptoms ofCAD include, e.g., pain, fever, pallor, anemia, reduced blood flow tothe extremities (e.g., with gangrene), and renal disease or acute renalfailure. In some embodiments, the symptoms can occur following exposureto cold temperatures.

From the above it will be clear that subjects “suspected of having acomplement-associated disorder” are not all the subjects within aspecies of interest.

In some embodiments, the methods can include identifying the subject asone having, suspected of having, or at risk for developing, acomplement-associated disorder in a subject. Suitable methods foridentifying the subject are known in the art. For example, suitablemethods (e.g., sequencing techniques or use of microarrays) fordetermining whether a human subject has a DDD-associated mutation in aCFH, CFHR5, or C3 gene are described in, e.g., Licht et al. (2006)KidneyInt 70:42-50; Zipfel et al. (2006), supra; Ault et al. (1997) J BiolChem 272:25168-75; Abrera-Abeleda et al. (2006) J Med Genet 43:582-589;Poznansky et al. (1989) J Immunol 143:1254-1258; Jansen et al. (1998)Kidney Int 53:331-349; and Hegasy et al. (2002) Am J Pathol161:2027-2034. Methods for detecting the presence of characteristicDDD-associated electron-dense deposits are also well known in the art.For example, a medical practitioner can obtain a tissue biopsy from thekidney of a patient and subject the tissue to electron microscopy. Themedical practitioner may also examine the tissue by immunofluorescenceto detect the presence of C3 using an anti-C3 antibody and/or lightmicroscopy to determine if there is membranoproliferativeglomerulonephritis. See, e.g., Walker et al. (2007) Mod Pathol20:605-616 and Habib et al. (1975) Kidney Int 7:204-215. In someembodiments, the identification of a subject as one having DDD caninclude assaying a blood sample for the presence of C3NeF. Methods fordetecting the presence of C3NeF in blood are described in, e.g.,Schwertz et al. (2001) Pediatr Allergy Immunol 12:166-172.

In some embodiments, the medical practitioner can determine whetherthere is increased complement activation in a subject's serum. Indiciaof increased complement activation include, e.g., a reduction in CH50, adecrease in C3, and an increase in C3dg/C3d. See, e.g., Appel et al.(2005), supra. In some embodiments, a medical practitioner can examine asubject's eye for evidence of the development of drusen and/or othervisual pathologies such as AMD. For example, a medical practitioner canuse tests of retinal function such as, but not limited to, darkadaptation, electroretinography, and electrooculography (see, e.g.,Colville et al. (2003) Am J Kidney Dis 42:E2-5).

Methods for identifying a subject as one having, suspected of having, orat risk for developing, TTP are also known in the art. For example,Miyata et al. describe a variety of assays for measuring ADAMTS13activity in a biological sample obtained from a subject (Curr OpinHematol (2007) 14(3):277-283). Suitable ADAMTS13 activity assays, aswell as phenotypically normal ranges of ADAMTS13 activity in a humansubject, are described in, e.g., Tsai (2003) J Am Soc Nephrol14:1072-1081; Furlan et al. (1998) New Engl J Med 339:1578-1584;Matsumoto et al. (2004) Blood 103:1305-1310; and Mori et al. (2002)Transfusion 42:572-580. Methods for detecting the presence of inhibitorsof ADAMTS13 (e.g., autoantibodies that bind to ADAMTS13) in a biologicalsample obtained from a subject are known in the art. For example, aserum sample from a patient can be mixed with a serum sample from asubject without TTP to detect the presence of anti-ADAMTS13 antibodies.In another example, immunoglobulin protein can be isolated from patientserum and used in in vitro ADAMTS13 activity assays to determine if ananti-ADAMTS13 antibody is present. See, e.g., Dong et al. (2008) Am JHematol 83(10):815-817. In some embodiments, risk of developing TTP canbe determined by assessing whether a patient carries one or moremutations in the ADAMTS13 gene. Suitable methods (e.g., nucleic acidarrays or DNA sequencing) for detecting a mutation in the ADAMTS13 geneare known in the art and described in, e.g., Levy et al., supra; Kokameet al., supra; Licht et al., supra; and Noris et al., supra.

In addition, methods for identifying a subject as one having, suspectedof having, or at risk for developing aHUS are known in the art. Forexample, laboratory tests can be performed to determine whether a humansubject has thrombocytopenia, microangiopathic hemolytic anemia, oracute renal insufficiency. Thrombocytopenia can be diagnosed by amedical professional as one or more of: (i) a platelet count that isless than 150,000/mm³ (e.g., less than 60,000/mm³); (ii) a reduction inplatelet survival time, reflecting enhanced platelet disruption in thecirculation; and (iii) giant platelets observed in a peripheral smear,which is consistent with secondary activation of thrombocytopoiesis.Microangiopathic hemolytic anemia can be diagnosed by a medicalprofessional as one or more of: (i) hemoglobin concentrations that areless than 10 mg/dL (e.g., less than 6.5 mg/dL); (ii) increased serumlactate dehydrogenase (LDH) concentrations (>460 U/L); (iii)hyperbilirubinemia, reticulocytosis, circulating free hemoglobin, andlow or undetectable haptoglobin concentrations; and (iv) the detectionof fragmented red blood cells (schistocytes) with the typical aspect ofburr or helmet cells in the peripheral smear together with a negativeCoombs test. (See, e.g., Kaplan et al. (1992) “Hemolytic Uremic Syndromeand Thrombotic Thrombocytopenic Purpura,” Informa Health Care (ISBN0824786637) and Zipfel (2005) “Complement and Kidney Disease,” Springer(ISBN 3764371668).)

A subject can also be identified as having aHUS by evaluating bloodconcentrations of C3 and C4 as a measure of complement activation ordysregulation. In addition, as is clear from the foregoing disclosure, asubject can be identified as having genetic aHUS by identifying thesubject as harboring one or more mutations in a gene associated withaHUS such as CFI, CFB, CFH, or MCP (supra). Suitable methods fordetecting a mutation in a gene include, e.g., DNA sequencing and nucleicacid array techniques. (See, e.g., Breslin et al. (2006) Clin Am SocNephrol 1:88-99 and Goicoechea de Jorge et al. (2007) Proc Natl Acad SciUSA 104:240-245.)

Methods for diagnosing a subject as one having, suspected of having, orat risk for developing, RA are also known in the art of medicine. Forexample, a medical practitioner can examine the small joints of thehands, wrists, feet, and knees to identify inflammation in a symmetricaldistribution. The practitioner may also perform a number of tests toexclude other types of joint inflammation including arthritis due toinfection or gout. In addition, rheumatoid arthritis is associated withabnormal antibodies in the blood circulation of afflicted patients. Forexample, an antibody referred to as “rheumatoid factor” is found inapproximately 80% of patients. In another example, anti-citrullineantibody is present in many patients with rheumatoid arthritis and thusit is useful in the diagnosis of rheumatoid arthritis when evaluatingpatients with unexplained joint inflammation. See, e.g., van Venrooij etal. (2008) Ann NY Acad Sci 1143:268-285 and Habib et al. (2007) ImmunolInvest 37(8):849-857. Another antibody called “the antinuclear antibody”(ANA) is also frequently found in patients with rheumatoid arthritis.See, e.g., Benucci et al. (2008) Clin Rheumatol 27(1):91-95; Julkunen etal. (2005) Scan J Rheumatol 34(2):122-124; and Miyawaki et al. (2005) JRheumatol 32(8):1488-1494.

A medical practitioner can also examine red blood cell sedimentationrate to help in diagnosing RA in a subject. The sedimentation rate canbe used as a crude measure of the inflammation of the joints and isusually faster during disease flares and slower during remissions.Another blood test that can be used to measure the degree ofinflammation present in the body is the C-reactive protein.

Furthermore, joint x-rays can also be used to diagnose a subject ashaving rheumatoid arthritis. As RA progresses, the x-rays can show bonyerosions typical of rheumatoid arthritis in the joints. Joint x-rays canalso be helpful in monitoring the progression of disease and jointdamage over time. Bone scanning, a radioactive test procedure, candemonstrate the inflamed joints.

Methods for identifying a subject as one having, suspected of having, orat risk for developing, HELLP are known in the art of medicine. Hallmarksymptoms of HELLP syndrome include hemolysis, elevated liver enzymes,and low platelet count. Thus, a variety of tests can be performed onblood from a subject to determine the level of hemolysis, theconcentration of any of a variety of liver enzymes, and the plateletlevel in the blood. For example, the presence of schistocytes and/orelevated free hemoglobin, bilirubin, or serum LDH levels is anindication of intravascular hemolysis. Routine laboratory testing can beused to determine the platelet count as well as the blood level of liverenzymes such as, but not limited to, aspartate aminotransferase (AST)and alanine transaminase (ALT). Suitable methods for identifying asubject as having HELLP syndrome are also described in, e.g., Sibai etal. (1993), supra; Martin et al. (1990), supra; Padden (1999), supra;and Gleicher and Buttino (1998) “Principles & Practice of MedicalTherapy in Pregnancy,” 3^(rd) Edition, Appleton & Lange (ISBN083857677X).

Methods for identifying a subject as having, suspected of having, or atrisk for developing PNH are known in the art of medicine. The laboratoryevaluation of hemolysis normally includes hematologic, serologic, andurine tests. Hematologic tests include an examination of the blood smearfor morphologic abnormalities of red blood cells (RBC), and themeasurement of the reticulocyte count in whole blood (to determine bonemarrow compensation for RBC loss). Serologic tests include lactatedehydrogenase (LDH; widely performed), and free hemoglobin (not widelyperformed) as a direct measure of hemolysis. LDH levels, in the absenceof tissue damage in other organs, can be useful in the diagnosis andmonitoring of patients with hemolysis. Other serologic tests includebilirubin or haptoglobin, as measures of breakdown products orscavenging reserve, respectively. Urine tests include bilirubin,hemosiderin, and free hemoglobin, and are generally used to measuregross severity of hemolysis and for differentiation of intravascular vs.extravascular etiologies of hemolysis rather than routine monitoring ofhemolysis. Further, RBC numbers, RBC hemoglobin, and hematocrit aregenerally performed to determine the extent of any accompanying anemia.

Suitable methods for identifying the subject as having MG can bequalitative or quantitative. For example, a medical practitioner canexamine the status of a subject's motor functions using a physicalexamination. Other qualitative tests include, e.g., an ice-pack test,wherein an ice pack is applied to a subject's eye (in a case of ocularMG) to determine if one or more symptoms (e.g., ptosis) are improved bycold (see, e.g., Sethi et al. (1987) Neurology 37(8):1383-1385). Othertests include, e.g., the “sleep test,” which is based on the tendencyfor MG symptoms to improve following rest. In some embodiments,quantitative or semi-quantitative tests can be employed by a medicalpractitioner to determine if a subject has, is suspected of having, oris at risk for developing, MG. For example, a medical practitioner canperform a test to detect the presence or amount of MG-associatedautoantibodies in a serum sample obtained from a subject. MG-associatedautoantibodies include, e.g., antibodies that bind to, and modulate theactivity of, acetylcholine receptor (AChR), muscle-specific receptortyrosine kinase (MuSK), and/or striational protein. (See, e.g.,Conti-Fine et al. (2006), supra). Suitable assays useful for detectingthe presence or amount of an MG-associated antibody in a biologicalsample are known in the art and described in, e.g., Hoch et al. (2001)Nat Med 7:365-368; Vincent et al. (2004) Semin Neurol 24:125-133;McConville et al. (2004) Ann Neurol 55:580-584; Boneva et al. (2006) JNeuroimmunol 177:119-131; and Romi et al. (2005) Arch Neurol 62:442-446.

Additional methods for diagnosing MG include, e.g., electrodiagnostictests (e.g., single-fiber electromyography) and the Tensilon (oredrophonium) test, which involves injecting a subject with theacetylcholinesterase inhibitor edrophonium and monitoring the subjectfor an improvement in one or more symptoms. See, e.g., Pascuzzi (2003)Semin Neurol 23(1):83-88; Katirji et al. (2002) Neurol Clin 20:557-586;and “Guidelines in Electrodiagnostic Medicine. American Association ofElectrodiagnostic Medicine,” Muscle Nerve 15:229-253.

A subject can be identified as having CAD using an assay to detect thepresence or amount (titer) of agglutinating autoantibodies that bind tothe I antigen on red blood cells. The antibodies can be monoclonal(e.g., monoclonal IgM or IgA) or polyclonal. Suitable methods fordetecting these antibodies are described in, e.g., Christenson and Dacie(1957) Br J Haematol 3:153-164 and Christenson et al. (1957) Br JHaematol 3:262-275. A subject can also be diagnosed as having CAD usingone or more of a complete blood cell count (CBC), urinalysis,biochemical studies, and a Coombs test to test for hemolysis in blood.For example, biochemical studies can be used to detect elevated lactasedehydrogenase levels, elevated unconjugated bilirubin levels, lowhaptoglobin levels, and/or the presence of free plasma hemoglobin, allof which can be indicative of acute hemolysis. Other tests that can beused to detect CAD include detecting complement levels in the serum. Forexample, due to consumption during the acute phase of hemolysis,measured plasma complement levels (e.g., C2, C3, and C4) are decreasedin CAD.

Typical (or infectious) HUS, unlike aHUS, is often identifiable by aprodrome of diarrhea, often bloody in nature, which results frominfection with a shiga-toxin producing microorganism. A subject can beidentified as having typical HUS when shiga toxins and/or serumantibodies against shiga toxin or LPS are detected in the stool of anindividual. Suitable methods for testing for anti-shiga toxin antibodiesor LPS are known in the art. For example, methods for detectingantibodies that bind to shiga toxins Stx1 and Stx2 or LPS in humans aredescribed in, e.g., Ludwig et al. (2001) J Clin Microbiol39(6):2272-2279.

In some embodiments, a high concentration antibody solution describedherein can be administered to a subject as a monotherapy. Alternatively,as described above, the solution can be administered to a subject as acombination therapy with another treatment, e.g., another treatment forDDD, TTP, wet or dry AMD, aHUS, PNH, RA, HELLP, MG, CAD, CAPS, tHUS, orany other complement-associated disorder known in the art or describedherein. For example, the combination therapy can include administeringto the subject (e.g., a human patient) one or more additional agents(e.g., anti-coagulants, anti-hypertensives, or corticosteroids) thatprovide a therapeutic benefit to the subject who has, or is at risk ofdeveloping, DDD. In some embodiments, the combination therapy caninclude administering to the subject (e.g., a human patient) by way of ahigh concentration antibody solution an anti-C5 antibody and animmunosuppressive agent such as Remicade® for use in treating RA. Insome embodiments, a high concentration antibody solution and the one ormore additional active agents are administered at the same time. Inother embodiments, a high concentration antibody solution isadministered first in time and the one or more additional active agentsare administered second in time. In some embodiments, the one or moreadditional active agents are administered first in time and highconcentration antibody solution is administered second in time.

An anti-C5 antibody described herein can replace or augment a previouslyor currently administered therapy. For example, upon treating with ananti-C5 antibody, administration of the one or more additional activeagents can cease or diminish, e.g., be administered at lower levels. Insome embodiments, administration of the previous therapy can bemaintained. In some embodiments, a previous therapy will be maintaineduntil the level of the anti-C5 antibody reaches a level sufficient toprovide a therapeutic effect. The two therapies can be administered incombination.

Monitoring a subject (e.g., a human patient) for an improvement in acomplement-associated disorder, as defined herein, means evaluating thesubject for a change in a disease parameter, e.g., an improvement in oneor more symptoms of the disease (e.g., an improvement in one or moresymptoms of a pulmonary disorder). Such symptoms include any of thesymptoms of complement-associated disorders known in the art and/ordescribed herein. In some embodiments, the evaluation is performed atleast 1 hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or atleast 1 day, 2 days, 4 days, 10 days, 13 days, 20 days or more, or atleast 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or more,after an administration. The subject can be evaluated in one or more ofthe following periods: prior to beginning of treatment; during thetreatment; or after one or more elements of the treatment have beenadministered. Evaluating can include evaluating the need for furthertreatment, e.g., evaluating whether a dosage, frequency ofadministration, or duration of treatment should be altered. It can alsoinclude evaluating the need to add or drop a selected therapeuticmodality, e.g., adding or dropping any of the treatments for any of thecomplement-associated disorders described herein.

Therapeutic Kits

The disclosure also features therapeutic kits containing, among otherthings, one or more of the high concentration solutions describedherein. The therapeutic kits can contain, e.g., a suitable means fordelivery of one or more solutions to a patient in need thereof, e.g., apatient afflicted with, suspected of having, or at risk for developing,a complement-associated disorder such as AMD (e.g., wet or dry AMD), adiabetes-associated ocular disorder, central retinal vein occlusion, RA,asthma, or any of the additional complement-associated disordersdescribed herein. In some embodiments, the means is suitable forinvasive (e.g., intravascular (e.g., intravenous), subcutaneous,intraarticular, intraocular, intravitreal, or intramuscular) delivery ofthe solution to a patient. In some embodiments, the means is suitablefor subcutaneous delivery of the antibody or antigen-binding fragmentthereof to the subject. For example, the means can be a syringe or anosmotic pump. In some embodiments, the solution contained in the kit canbe formulated as an eye drop, the means being an eye dropper. In someembodiments, the kit contains a means that is pre-loaded with thesolution to be administered. For example, a therapeutic kit can containa syringe pre-filled with an aqueous solution (e.g., a pen devicecontaining the solution) described herein or the kit can contain a pump(e.g., an osmotic pump) and one or more disposable cassettes configuredfor use with the pump, the cassettes pre-filled with an aqueous solutiondescribed herein. In another example, the kit can contain a transscleralor implantable delivery device (e.g., a plug) that is pre-filled with(or otherwise contains) a high concentration solution described herein.

In some embodiments, the means for delivering the high concentrationsolution is a pen device for drug delivery.

In some embodiments, the means can be suitable for administration of ahigh concentration antibody solution described herein to the eye of apatient afflicted with a complement-associated disorder of the eye suchas AMD. The means can be, e.g., a syringe, a transscleral patch, or evena contact lens containing or soaked in the solution. The means can, insome embodiments, be an eye dropper, wherein the solution is formulatedfor such administration. The means can also be, e.g., a contact lenscase in embodiments in which, e.g., the solution is formulated as partof a contact lens hydrating, cleaning, or soaking solution. Suchtherapeutic kits can also include, e.g., one or more additionaltherapeutic agents for use in treating complement-associated disorder ofthe eye. The therapeutic agents can be, e.g., bevacizumab or the Fabfragment of bevacizumab, ranibizumab, both sold by RochePharmaceuticals, Inc., pegaptanib sodium (Mucogen®; Pfizer, Inc.), andverteporfin (Visudyne®; Novartis). Such a kit can also, optionally,include instructions for administering a solution described herein to apatient.

In some embodiments, the means can be suitable for intraarticularadministration of a solution described herein to a patient in needthereof, e.g., a patient afflicted with complement-associated disorderaffecting the joints such as RA. The means can be, e.g., a syringe or adouble-barreled syringe. See, e.g., U.S. Pat. Nos. 6,065,645 and6,698,622. A double-barreled syringe is useful for administering to ajoint two different compositions with only one injection. Two separatesyringes may be incorporated for use in administering the therapeuticwhile drawing off knee fluid for analysis (tapping) in a push-pullfashion. Additional therapeutic agents that can be administered with thehigh concentration antibody solutions described herein in conjunctionwith the double-barreled syringe, or which can otherwise be generallyincluded in the therapeutic kits described herein, include, e.g.,NSAIDs, corticosteroids, methotrexate, hydroxychloroquine, anti-TNFagents such as etanercept and infliximab, a B cell depleting agent suchas rituximab, an interleukin-1 antagonist, or a T cell costimulatoryblocking agent such as abatacept. Such a kit can also, optionally,include instructions for administering a solution described herein to apatient.

In some embodiments, the means is suitable for intrapulmonary deliveryof the solutions to a subject, e.g., for use in treatment or preventionof a complement-associated pulmonary disorder such as, but not limitedto, COPD or asthma. Accordingly, the means can be, e.g., an oral ornasal inhaler (see above). The inhaler can be, e.g., a metered doseinhaler (MDI) or a nebulizer. Such a kit can also, optionally, includeinstructions for administering (e.g., self-administration of) theanti-C5a antibody or antigen-binding fragment thereof to a subject. Thetherapeutic kits are designed for use in treating or preventing acomplement-associated pulmonary disorder and can include one or moreadditional active agents including, but not limited to, another antibodytherapeutic (e.g., an anti-IgE antibody, an anti-IL-4 antibody, or ananti-IL-5 antibody), a small molecule anti-IgE inhibitor (e.g.,montelukast sodium), a sympathomimetic (e.g., albuterol), an antibiotic(e.g., tobramycin), a deoxyribonuclease (e.g., Pulmozyme®), ananticholinergic drug (e.g., ipratropium bromide), a corticosteroid(e.g., dexamethasone), a β-adrenoreceptor agonist, a leukotrieneinhibitor (e.g., zileuton), a 5-lipoxygenase inhibitor, aphosphodiesterase (PDE) inhibitor, a CD23 antagonist, an IL-13antagonist, a cytokine release inhibitor, a histamine H1 receptorantagonist, an anti-histamine, an anti-inflammatory agent (e.g.,cromolyn sodium or any other anti-inflammatory agent known in the art ordescribed herein), or a histamine release inhibitor.

The following examples are intended to illustrate, not limit, theinvention.

EXAMPLES Example 1 Process for Formulating and Concentrating Solutionsof Eculizumab

Several high concentration formulations of the anti-C5 antibody,eculizumab, were prepared as follows.

Materials and Methods

Instrumentation

The formulation process utilized a Millipore Pellicon® XL tangentialflow filter (TFF), Biomax™ 50K Polyethersulfone membrane having a 50 cm²surface area. Also used was a Millipore Sterivex 0.22 micron filter unit(catalogue number SVGV010RS).

Reagents

The formulation process also utilized a number of buffers as follows:(a) Formulation Buffer: 20 mM histidine, 50 mM serine, 2.5% sorbitol,1.5% mannitol, pH 7.4; (b) Phosphate Buffer: 20 mM sodium phosphate, 80mM NaCl, pH 6.4; (c) Regeneration Buffer: 0.5 M sodium hydroxide; and(d) Storage Buffer: 0.1 M sodium hydroxide. Also used was a buffercontaining eculizumab in the above-described Phosphate Buffer.

Formulation

The tangential flow filter (TFF) was prepared by washing from it thestorage buffer using 500 mL of deionized water at a feed flow rate (FR)of 50 mL/minute. The permeate outlet was left open during this process.The TFF was equilibrated using 100 mL of the Phosphate Buffer at a feedflow rate of 50 mL/minute with the permeate outlet open. All of thesesteps were performed at room temperature.

For all subsequent steps, the pressure was maintained at 40 psi byadjusting the permeate outlet flow rate with a clamp. Eculizumab,initially present at approximately 10 mg/mL in Phosphate Buffer (asdescribed above), was concentrated to 50 mg/mL (for the first run and 40mg/ml for the second run) at a feed flow rate of 50 mL/minute. Theconcentrated solution was then diafiltered with six equivalent volumes,for the first run, or four equivalent volumes, for the second run, ofthe Formulation Buffer. Concentration was continued by graduallyreducing the feed flow rate to maintain column pressure at 40 psi, untilthe feed flow rate reached 2 mL/minute.

Recovery Method

To recover the concentrated eculizumab solution from the column, thepermeate outlet was closed and the antibody allowed to circulate forfive minutes. The TFF was then flushed out with air, while the permeateoutlet was closed. The volume of the recovered solution was measured andrecorded. A sample of the high concentration antibody solution wasfiltered through a 0.22 micron Sterivex filter, diluted 1:100 informulation buffer. The concentration of the antibody in the dilutedsample was determined by measuring A₂₈₀ and using an extinctioncoefficient of 1.46.

Results

The first run process, described above, required 5.3 hours to complete.A detailed description of the physical and chemical parameters of theTFF flow-through (permeate) and retained (retentate) fractions by timeis shown in Table 1.

TABLE 1 Est. Feed Permeate Retentate Permeate Permeate Retentate TimeTime Pressure FR FR Volume Volume Conc. Conc. (min) (hrs) (psi) (ml/min)(ml/min) (ml) (ml) (mg/ml) (mg/ml) Details 0 0.0 40 50 0.0 550 0 0.0510.3 x^(a) 24 0.4 40 50 7.5 370 180 0.05 15 52 0.9 40 50 6.1 200 3500.05 29 68 1.1 40 50 4.7 120 425 0.05 46 Diafiltr. 104 1.7 40 50 3.9 120140 0.05 47 Diafiltr. 118 2.0 40 50 3.6 120 190 0.05 47 Diafiltr. 1262.1 40 50 3.8 120 220 0.05 47 Diafiltr. 146 2.4 40 50 4.0 120 300 0.0547 Diafiltr. 226 3.8 40 50 3.8 120 600 0.05 47 Diafiltr. 241 4.0 40 472.5 70 38 0.05 81 247 4.1 40 41 1.7 60 48 0.05 95 254 4.2 40 35 1.0 5355 0.05 107 261 4.4 40 27 0.7 48 60 0.05 116 x^(b) 275 4.6 40 15 0.7 3870 0.05 150 294 4.9 40 6 0.3 33 75 0.05 172 318 5.3 35 2 0.2 28 80 0.05186 x^(c) x^(a): 5.7 g at 100% purity; actual measured retentateconcentration. x^(b): Recovered 98% at 100% purity; actual measuredretentate concentration. x^(c): Recovered 75% at 100% purity; actualmeasured retentate concentration. “Diafiltr.” refers to diafiltration.“FR” refers to flow rate. The “conc.” refers to the concentration ofeculizumab in each respective fraction (permeate or retentate).The initial concentration of eculizumab in the Phosphate Buffer wasapproximately 10 mg/mL (10.3 mg/mL). Following buffer exchange anddiafiltration, the concentration of the solution was initially increasedto 116 mg/mL with 98% recovery (100% purity) of the antibody startingmaterial. Further concentration to 186 mg/mL resulted in a 75% recovery(at 100% purity) of the antibody starting material.

The second run process, described above, required 8.8 hours to complete.A detailed description of the physical and chemical parameters of theTFF flow-through (permeate) and retained (retentate) fractions by timeis shown in Table 2.

TABLE 2 Est. Feed Permeate Retentate Permeate Permeate Retentate TimeTime Pressure FR FR Volume Volume Conc. Conc. (min) (hrs) (psi) (ml/min)(ml/min) (ml) (ml) (mg/ml) (mg/ml) Details 0 0.0 40 50 0.0 620 0 0.06 8x^(a) 10 0.2 40 50 7.5 545 75 0.06 10 33 0.6 40 50 6.5 395 225 0.06 1450 0.8 40 50 5.6 300 320 0.06 18 70 1.2 40 50 4.8 205 415 0.06 26 85 1.440 50 3.7 150 470 0.06 35 137 2.3 40 50 3.8 150 200 0.06 37 Diafiltr.237 4.0 40 50 3.0 150 500 0.06 37 Diafiltr. 303 5.1 40 50 3.0 150 7000.06 37 Diafiltr. 367 6.1 40 50 3.1 150 900 0.06 37 Diafiltr. 377 6.3 4050 2.6 119 26 0.06 45 382 6.4 40 50 2.8 105 40 0.06 51 388 6.5 40 50 2.093 52 0.06 58 397 6.6 40 50 1.8 77 68 0.06 70 410 6.8 40 50 1.1 63 820.06 86 422 7.0 40 40 0.8 53 92 0.06 101 431 7.2 40 30 0.7 47 98 0.06114 444 7.4 40 21 0.5 41 104 0.06 131 451 7.5 40 16 0.3 39 106 0.06 138456 7.6 40 15 0.4 37 108 0.06 145 468 7.8 40 9 0.2 35 110 0.06 151 x^(b)489 8.2 40 5 0.1 33 112 0.06 163 527 8.8 40 2 0.2 25 120 0.06 208 x^(c)x^(a): 5.37 g at 100% purity; actual measured retentate concentration.x^(b): Recovered 98% at 100% purity; actual measured retentateconcentration. x^(c): Recovered 85% at 100% purity; actual measuredretentate concentration. “Diafiltr.” refers to diafiltration. “FR”refers to flow rate. The “conc.” refers to the concentration ofeculizumab in each respective fraction (permeate or retentate).The initial concentration of eculizumab in the Phosphate Buffer wasapproximately 10 mg/mL (8.4 mg/mL). Following buffer exchange anddiafiltration, the concentration of the solution was initially increasedto 151 mg/mL with 98% recovery (100% purity as determined by SEC-HPLC)of the antibody starting material. Further concentration to 208 mg/mLresulted in an 85% recovery (at 100% purity) of the antibody startingmaterial.

From the results of the first and second run, diafiltering at 40 mg/mLnot only improved the final recovery by 10%, but also allowed for theproduction of high concentration solution having an even higher finalconcentration of eculizumab (208 mg/mL).

Example 2 Production of Additional Exemplary Eculizumab Formulations

Two additional formulations were developed and evaluated as to theirability to support high concentration solutions of eculizumab. One ofthe formulations was a histidine/serine/sorbitol/mannitol (HSSM)formulation and the other, a histidine/trehalose/Tween®-20 (HTT)formulation (see below). Two concentrations of eculizumab, approximately30 mg/mL and approximately 100 mg/mL, were evaluated in each formulationbuffer. A third, phosphate-based buffer was also evaluated. A detaileddescription of the five different antibody solutions (solutions I to V)evaluated is set forth below.

I. 105 mg/mL eculizumab;

-   -   20 mM histidine HCl;    -   50 mM serine;    -   3% sorbitol; and    -   1.5% mannitol; at pH 7.0.

II. 30 mg/mL eculizumab;

-   -   20 mM histidine HCl;    -   50 mM serine;    -   3% sorbitol; and    -   1.5% mannitol; at pH 7.0.

III. 105 mg/mL eculizumab;

-   -   10 mM histidine HCl;    -   10% alpha-trehalose dihydrate; and    -   0.01% polysorbate 20; at pH 7.0.

IV. 30.2 mg/mL eculizumab;

-   -   10 mM histidine HCl;    -   10% alpha-trehalose dehydrate; and    -   0.01% polysorbate 20; at pH 7.0.

V. 10 mg/mL eculizumab;

-   -   10 mM sodium phosphate;    -   150 mM sodium chloride; and    -   0.02% polysorbate 80; at pH 7.0.

Solutions I-V were prepared by way of concentration and formulation asdescribed above in Example 1, or with only routine and minormodifications to the procedures. Briefly, to prepare solutions I and II,a 10 mg/mL solution of eculizumab in a phosphate-based buffer wasconcentrated to 30 mg/mL using a TFF. Next, the 30 mg/mL concentrate wassubjected to six rounds of diafiltration (as described above inExample 1) into the HSSM formulation (20 mM histidine, 50 mM serine, 3%sorbitol, and 1.5% mannitol, at pH 7.0) to thereby produce solution II.A portion of solution II was further concentrated as described inExample 1 to 100 mg/mL, to produce solution I (the retentate).

Solution IV was prepared by diafiltration of the 30 mg/mL,phosphate-based eculizumab solution in the HTT buffer (10 mM histidineHCl; 10% alpha-trehalose dihydrate and 0.01% polysorbate 20, at pH 7.0),followed by the addition of Tween 20 to 0.01%. To prepare solution III,the 30 mg/mL, phosphate-based eculizumab solution was diafiltered in HTTbuffer and then further concentrated using the TFF as described inExample 1. Tween 20 was added to the retentate to a concentration of0.01%. A flow chart depicting the steps for formulation of the fivesolutions is shown in FIG. 1. Each of the solutions was passed through a0.22 μM filter.

Example 3 Stability of an Anti-C5 Antibody Formulated at HighConcentration

A series of experiments was performed to evaluate the structural andfunctional stability of eculizumab formulated at high concentrations inaqueous solution (as prepared in Example 2). Sample aliquots of 2 mLwere stored at −20° C., 2-8° C., and 37° C., and then evaluated atspecified time intervals (e.g., one month, two months, three months, sixmonths, nine months, 12 months, 18 months, and 24 months). The solutionswere subjected to a number of chemical evaluations: appearance (visualinspection), osmolality, concentration (using a UV spectrophotometer),purity (by size exclusion chromatography-HPLC), isoelectric focusing(IEF), and sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). The functional stability of the antibody in each solutionwas tested using a human C5-binding assay. The results of each of theevaluations are provided below in the following tables (Roman numerals Ito V in the tables correspond to the five named solutions above).

Methods and Results

A. Appearance (Visual Color, Clarity, Particles)

The appearance of each solution (under the varied storage conditions)was evaluated visually by observation of the vial against both a whiteand a black background. The test was performed at the intervals recitedin the following tables for samples maintained at 2-8° C., −20° C., and37° C. All solutions stored at 2-8° C. were found to be clear,colorless, and particulate free even after 24 months of storage.

All solutions stored at −20° C. were found to be clear, colorless, andparticulate free at one month of storage. Solution V was not testedbeyond 1 month, but solutions I to IV were clear, colorless, andparticulate free for up to 6 months of storage. At 12 months, solutionII contained small white particles. At 24 months solution I alsocontained small white particles. Solutions III and IV remained clear,colorless, and particulate free for at least 24 months.

All solutions stored at 37° C. were found to be clear, colorless, andparticulate free for at least 1 month. After two months of storage at37° C., solutions I and III appeared pale yellow in color. After threemonths of storage at 37° C., solutions I, II, III, and IV all appearedpale yellow in color and remained unchanged through six months.

B. Osmolality

The osmolality of each solution was measured using freezing pointdepression. The test was performed at the time intervals recited in thetables below for samples stored at 2-8° C., −20° C., and 37° C. Sampleswere tested in triplicate and the value reported herein is the mean ofthe three results.

All solutions stored at 2-8° C. had initial (T₀) osmolalities rangingfrom 299 to 365 mOsm/kg. After 24 months of storage at 2-8° C., theosmolality for each solution showed slight fluctuations, which werewithin the error of the method. The measured osmolalities for solutionsI, II, and IV stored at 2-8° C. remained within ±15% of the initialmeasured osmolality, which is typically the osmolality specification forsolutions in the earliest stage of development.

All solutions stored at −20° C. had initially-measured osmolalitiesranging from 299 to 365 mOsm/kg. The osmolality for each solution storedup to 24 months at −20° C. showed slight fluctuations, which are withinthe error of the method. The osmolalities for all solutions remainedwithin ±15% of the initially-measured osmolalities.

All solutions stored at 37° C. had initial (T₀) osmolalities rangingfrom 299 to 365 mOsm/kg. The osmolality of solutions I, II, and IVshowed slight fluctuations during the first six months measurement, allof which were within the error of the method. The osmolalities for allsolutions remained within ±15% of the initial osmolality. Solution III,however, had a measured osmolality of 863 mOsm/kg at 6 months, wellabove ±15% of the initial osmolality. While the disclosure is not boundby any particular theory or mechanism of action, it is believed that thesolution by six months storage at 37° C. had undergone significantdegradation, which resulted in an aberrant measurement at this timepoint.

C. Protein Concentration

Absorbance at 280 nm was used to determine the protein concentration ineach test sample using an extinction coefficient of 1.46. The testsample was diluted to give an absorbance reading in the linear range ofthe assay (0.2 to 1.0 absorbance units). The absorbance of triplicatesamples was measured by one operator, and then repeated independently bya second operator. The value reported is determined from the absorbancemean of the six measurements and the applied extinction coefficient.

D. Purity by HPLC Gel Permeation

The relative percents of monomeric IgG, aggregate, and fragments of theanti-C5 antibody were determined using SEC-HPLC (also referred to as gelpermeation (GP)-HPLC). Test samples were injected onto a TSKgel G3000SWXL column (Sigma-Aldrich) equilibrated with phosphate buffered saline(PBS), pH 7.0. The isocratic elution of the proteins is accomplishedwith a 20 minute run using the PBS, pH 7.0, at a flow rate of 1.0mL/minute. Protein peaks were monitored by spectrophotometry at awavelength of 214 nm and the percent purity of the monomeric IgG isexpressed as a percentage of the total integrated peak area. Detectionof the larger mass multimers was by observation of peaks eluting priorto the monomer peak. A measurement was made at each of the intervalsrecited in the following tables for samples stored at 2-8° C., −20° C.,and 37° C.

All solutions stored at 2-8° C. had an initially-measured (T₀) purity of99.1% monomer or greater. The purity for most of the solutions at up tosix months of storage at 2-8° C. showed slight fluctuations equal to thevariability of the method throughout the study. The purity for allsolutions remained equal to or greater than 98.0% for up to 24 months ofstorage. The stability profile of solution II (in the so-called “HSSM”formulation) most closely resembled the profile of the control (SolutionIV—10 mg/mL in 10 mM sodium phosphate, 150 mM sodium chloride, 0.02%polysorbate 80, pH 7.0).

All solutions stored at −20° C. had a purity of 99.1% monomer or greaterat T=0. From T=0 through T=24 months, the purity for most of thesolutions showed slight fluctuations equal to the variability of themethod throughout the study. The purity of Solutions 1 and 2 remainedvirtually unchanged through T=24 months. The purity of Solution 3remained above 98.7% through 12 months, and then dropped to 97.3% at 24months. Solution 4 had a purity of ≧98.5% through 24 months. Solutions1, 2, and 4 all remained at above 98.0% through 24 months. Solution 3remained above 95.0% through 24 months.

All solutions stored at 37° C. had a purity of 99.1% monomer or greaterat T=0. Beginning at the 1 month time point, significant increases inaggregate and fragments were seen in all solutions. The purity ofsolutions I, II, and III remained above 90.5% through six months.Solution IV had a purity of ≧97.4% through six months.

Solutions III and IV contained detectable amounts of fragment at T₀ (0.3and 0.7%, respectively) which then increased in all storage conditionsduring the study.

E. SDS-PAGE (Non-Reduced Coomassie)

Samples of the stored solutions were denatured by heating in thepresence of sodium dodecyl sulfate (SDS). The polypeptides present inthe samples were separated according to molecular size byelectrophoresis through a gradient 4% to 20% w/v pre-cast Tris-glycineSDS-polyacrylamide gel. The proteins within the gel were visualized bystaining the gel with Coomassie Blue.

Polypeptide bands within the gels were quantified using laserdensitometry. The limit of quantitation of the staining procedure wasapproximately 0.08 μg/polypeptide band. That is, when an 8 μg testsample was applied, the limit of quantitation of a single discreteimpurity is equivalent to approximately 1.0% of the total protein. Thereproducibility of the method expressed as a coefficient of variation isapproximately 1.8%. The test was performed at the intervals in thefollowing tables (below) for samples stored at 2-8° C., −20° C., and 37°C.

All solutions stored at 2-8° C. had an initially-measured (T₀) relativepercentage of at least 90% IgG. The percent IgG for all solutions storedat 2-8° C. was ≧90% at 24 months. All solutions stored at −20° C. alsohad an initially-measured relative percentage of 90% IgG. The percentIgG for solutions I and IV was ≧90% at 24 months. Solutions II and IIIhad percentage of IgG of 89% and 88%, respectively, at 24 months. As thepercentage of IgG at T₀ was 90%, 88% and 89% were within the error ofthe method and did not necessarily represent a significant change inquality.

All solutions stored at 37° C. had a relative percent IgG of 90% at T₀.The percentage of IgG for solution IV remained ≧90% through 3 months ofstorage. Solution I contained 88% IgG at 3 months, which was within theerror of the method, and did not necessarily represent a significantchange in quality. At 6 months of storage at 37° C., all solutions were≦73% IgG, indicating significant degradation of the antibody.

F. SDS-PAGE (Reduced Coomassie)

Samples were prepared and analyzed as described under Section E“SDS-PAGE (Non-reduced Coomassie)”; however, the samples were furtherdenatured in the presence of 50 mM dithiothreitol (DTT) to disruptdisulfide bonds within the antibody structure. The test was performed atthe intervals in the following tables (below) for samples stored at 2-8°C., −20° C., and 37° C.

All solutions stored at 2-8° C. had a relative percent of IgG as heavyand light chain of 100% at T₀. The percent of IgG as heavy and lightchain for all of the solutions stored at 2-8° C. remained at 100%throughout the study. All solutions stored at −20° C. also had arelative percent of IgG as heavy and light chain of 100% throughout thestudy.

All solutions stored at 37° C. had a relative percent of IgG as heavyand light chain of 100% at T₀. At 1 month, the relative percentage ofIgG as heavy and light chain remained at 100% for solutions III and IV.The percentage of IgG as heavy and light chains for solution I fell to99% and for Solution II fell to 98% after one month of storage at 37° C.After two months of storage at 37° C., all solutions were ≦98.0% IgG asheavy and light chains. The percentage of IgG as heavy and light chainscontinued to fall through six months of storage at 37° C. for eachsolution, indicating significant degradation of the antibody.

G. Isoelectric Focusing

The isoelectric focusing studies used a flat bed electrophoresis system.Pre-cast agarose gels covering a pH range of 3.0 to 10.0 were employed.Samples of the stored samples were loaded onto the gel at apredetermined optimized load position along with commercially availablepI marker standards. Following focusing for a set number of volt-hours,separated charge variants were visualized by staining with a CoomassieBlue staining solution. The banding pattern of samples on the stainedgel was analyzed using laser densitometry. The pI of the separatedisoforms and the relative mass of each isoform (as a percentage of totalmass) was also determined. pIs are calculated by interpolation from astandard curve established by the pI marker standards. The relative massof each isoform as a percent of total mass is calculated from itsresponse relative to the total response of the sample load. Themeasurements were performed at the intervals in the following tables(below) for samples stored at 2-8° C., −20° C., and 37° C.

All solutions stored at 2-8° C. had banding patterns comparable to areference antibody standard and pI ranges of approximately 5.66 to 6.35at T₀. Minor variations in pI were seen throughout the study, which arewithin the variability of the method. After up to 12 months of storage,banding patterns of the antibody present in the samples were comparableto the reference antibody standard and the pI of all bands remainedbetween 5.48 and 6.60 for all of the solutions. Solutions I and II hadbanding patterns which remained comparable to the reference antibodymaterial and the pI of all bands between 5.48 and 6.60 through 24 monthsof storage at 2-8° C. Solutions III and IV did exhibit a change in thebanding pattern beginning at the 18 month time point. That is, the mostbasic band was not detected.

All solutions stored at −20° C. had banding patterns comparable to thereference antibody material and pI ranges of ˜5.66 to 6.35 at T₀. Minorvariations in pI were seen throughout the study, which are within thevariability of the method. Throughout 24 months of storage at −20° C.,banding patterns were comparable to the reference antibody material andthe pI of all bands was between 5.61 and 6.44 for all of the solutions.

All solutions stored at 37° C. had banding patterns comparable to thereference antibody and pI ranges of ˜5.66 to 6.35 at T=0. After up to 1month of storage, banding patterns were comparable to reference materialand the pI of all bands was between 5.45 and 6.43 for all of thesolutions. After two months of storage, smearing between the bandsappeared, an additional acidic band appeared, and the intensities of thebands diminished. No significant difference was seen between the foursolutions tested at this temperature condition. The appearance ofsmearing, development of more acidic bands, and changes in intensity ofthe main band continued for the duration of the 24 month study.

H. Potency by C5 Binding Assay

The C5 binding assay used to test the functional characteristics of thestored solutions was a quantitative immunoassay. A standard curve wasprepared from a reference anti-C5 antibody standard to includeconcentrations at 500, 250, 125, 62.5 and 31.3 binding units (BU)/mL. Afour-parameter fit was applied to the standard curve and test sampleresults were interpolated from the curve. Each sample was diluted andtested in triplicate at each of three dilutions predetermined to fallwithin the linear range of the assay. Results were averaged and observedtest results in units of BU/mL were divided by the product concentrationin mg/mL to obtain results in BU/mg. The measurements were performed atthe intervals in the following tables (below) for samples stored at 2-8°C., −20° C., and 37° C.

All solutions stored at 2-8° C. exhibited binding activity ranging from946,875 to 1,063,353 BU/mg at T₀. Throughout the 24 month study, thepurity for all of the tested solutions remained between 855,801 and1,194,123 BU/mg. At 12 months, solution I contained 1,306,497 BU/mg, butat 18 and 24 months solution I contained 1,013,876 and 920,747 BU/mg,respectively. All solutions stored at −20° C. contained 946,875 to1,063,353 BU/mg at T₀. Throughout the 24 month study, the activitypresent in all solutions stored at −20° C. remained between 778,672 and1,148,100 BU/mg.

All solutions stored at 37° C. exhibited initially-measured bindingactivity ranging from 946,875 to 1,063,353 BU/mg. After six months ofstorage at 37° C., solutions I and IV exhibiting activity of between827,206 and 1,202,435 BU/mg. Solutions II and III exhibited bindingactivity between 1,019,401 and 1,243,601 BU/mg after 3 months, whereas,at six months, the activity of the antibody maintained at 37° C. insolution II and III was 1,679,080 and 1,976,400 BU/mg respectively.Again, while the disclosure is not bound by any particular theory ormechanism of action, it is believed that the solution by six monthsstorage at 37° C. had undergone significant degradation, which resultedin an aberrant measurement at this time point.

TABLE 3 Appearance and Osmolality of Eculizumab Solutions at 2 to 8° C.for 24 Months Time Analytical Point 2-8° C. method (months) I II III IVV Appearance 0 Clear and Clear and Clear and Clear and Clear andcolorless, colorless, colorless, colorless, colorless, Particulate freeParticulate Particulate Particulate Particulate free free free free 1Clear and Clear and Clear and Clear and Clear and colorless, colorless,colorless, colorless, colorless, Particulate free ParticulateParticulate Particulate Particulate free free free free 2 Clear andClear and Clear and Clear and Clear and colorless, colorless, colorless,colorless, colorless, Particulate free Particulate ParticulateParticulate Particulate free free free free 3 Clear and Clear and Clearand Clear and Clear and colorless, colorless, colorless, colorless,colorless, Particulate free Particulate Particulate ParticulateParticulate free free free free 6 Clear and Clear and Clear and Clearand Clear and colorless, colorless, colorless, colorless, colorless,Particulate free Particulate Particulate Particulate Particulate freefree free free 9 Clear and Clear and Clear and Clear and Clear andcolorless, colorless, colorless, colorless, colorless, Particulate freeParticulate Particulate Particulate Particulate free free free free 12Clear and Clear and Clear and Clear and Clear and colorless, colorless,colorless, colorless, colorless, Particulate free ParticulateParticulate free Particulate Particulate free free free 18 Clear andClear and Clear and Clear and Clear and colorless, colorless, colorless,colorless, colorless, Particulate free Particulate Particulate freeParticulate Particulate free free free 24 Clear and Clear and Clear andClear and Clear and colorless, colorless, colorless, colorless,colorless, Particulate free Particulate Particulate free ParticulateParticulate free free free Osmolality 0 365 mOsm/kg 333 mOsm/kg 356mOsm/kg 313 mOsm/kg 299 mOsm/kg 1 2 3 6 367 mOsm/kg 336 mOsm/kg 364mOsm/kg 326 mOsm/kg 300 mOsm/kg 9 12 371 mOsm/kg 335 mOsm/kg 366 mOsm/kg310 mOsm/kg 305 mOsm/kg 18 366 mOsm/kg 337 mOsm/kg 358 mOsm/kg 324mOsm/kg 303 mOsm/kg 24 372 mOsm/kg 338 mOsm/kg 382 mOsm/kg 321 mOsm/kg306 mOsm/kg

TABLE 4 Protein Concentration and SEC-HPLC Evaluation of EculizumabSolutions Stored at 2 to 8° C. for 24 Months Time Analytical Point 2-8°C. method (months) I II III IV V Protein 0 105.9 mg/mL 29.3 mg/mL 107.6mg/mL 31.1 mg/mL 10.0 mg/mL Concentration 1 108.4 mg/mL 29.9 mg/mL 108.6mg/mL 31.2 mg/mL 10.2 mg/mL 2 104.0 mg/mL 29.1 mg/mL 101.6 mg/mL 30.0mg/mL 10.0 mg/mL 3 102.8 mg/mL 29.0 mg/mL 104.3 mg/mL 30.2 mg/mL  9.8mg/mL 6 104.0 mg/mL 29.1 mg/mL 103.1 mg/mL 30.4 mg/mL  9.9 mg/mL 9 110.0mg/mL 30.8 mg/mL 112.1 mg/mL 31.6 mg/mL 10.0 mg/mL 12 106.2 mg/mL 30.2mg/mL 109.8 mg/mL 31.3 mg/mL 10.1 mg/mL 18 108.7 mg/mL 30.6 mg/mL 110.9mg/mL 31.2 mg/mL 10.1 mg/mL 24 106.4 mg/mL 30.7 mg/mL 111.8 mg/mL 32.5mg/mL 10.3 mg/mL SEC-HPLC 0 0.3% 0.2% 0.3% 0.2% 0.2% aggregatesaggregates aggregates aggregates aggregates 99.7% 99.8% 99.5% 99.1%99.8% monomer monomer monomer monomer monomer 0% fragments 0% fragments0.3% 0.7% 0% fragments fragments fragments 1 0.4% 0.3% 0.4% 0.4% 0.3%aggregates aggregates aggregates aggregates aggregates 99.6% 99.7% 98.7%98.7% 99.7% monomer monomer monomer monomer monomer 0% fragments 0%fragments 0.9% 0.9% 0% fragments fragments fragments 2 0.5% 0.4% 0.4%0.3% 0.3% aggregates aggregates aggregates aggregates aggregates 99.5%99.6% 98.6% 98.6% 99.7% monomer monomer monomer monomer monomer 0%fragments 0% fragments 1.0% 1.2% 0% fragments fragments fragments 3 0.5%0.4% 0.3% 0.2% 0.3% aggregates aggregates aggregates aggregatesaggregates 99.5% 99.6% 98.7% 98.5% 99.7% monomer monomer monomer monomermonomer 0% fragments 0% fragments 1.0% 1.3% 0% fragments fragmentsfragments 6 0.6% 0.4% 0.4% 0.3% 0.3% aggregates aggregates aggregatesaggregates aggregates 99.4% 99.6% 98.8% 98.8% 99.6% monomer monomermonomer monomer monomer 0% fragments 0% fragments 0.7% 1.0% 0.1%fragments fragments fragments 9 0.9% 0.3% 0.5% 0.3% 0.4% aggregatesaggregates aggregates aggregates aggregates 99.2% 99.7% 99.3% 99.4%99.7% monomer monomer monomer monomer monomer 0% fragments 0% fragments0.2% 0.3% 0% fragments fragments fragments 12 1.3% 0.7% 0.5% 0.4% 0.6%aggregates aggregates aggregates aggregates aggregates 98.7% 99.3% 98.9%98.8% 99.5% monomer monomer monomer monomer monomer 0% fragments 0%fragments 0.6% 0.9% 0% fragments fragments fragments 18 1.6% 0.8% 0.6%0.3% 0.5% aggregates aggregates aggregates aggregates aggregates 98.4%99.2% 99.2% 99.3% 99.5% monomer monomer monomer monomer monomer 0%fragments 0% fragments 0.2% 0.3% 0% fragments fragments fragments 242.0% 0.9% 0.7% 0.3% 0.6% aggregates aggregates aggregates aggregatesaggregates 98.0% 99.1% 99.1% 99.4% 99.4% monomer monomer monomer monomermonomer 0% fragments 0% fragments 0.2% 0.3% 0% fragments fragmentsfragments

TABLE 5 SDS-PAGE Analysis of Eculizumab Solutions Stored at 2 to 8° C.for 24 Months Time Analytical Point 2-8° C. method (months) I II III IVV SDS-PAGE 0 90% IgG 90% IgG 90% IgG 90% IgG 90% IgG Non-reduced 1 92%IgG 92% IgG 92% IgG 92% IgG 92% IgG 2 91% IgG 91% IgG 92% IgG 90% IgG93% IgG 3 92% IgG 92% IgG 92% IgG 92% IgG 91% IgG 6 89% IgG 89% IgG 90%IgG 90% IgG 90% IgG 9 91% IgG 91% IgG 91% IgG 91% IgG 90% IgG 12 90% IgG90% IgG 90% IgG 90% IgG 91% IgG 18 90% IgG 90% IgG 91% IgG 90% IgG 91%IgG 24 91% IgG 90% IgG 91% IgG 90% IgG 91% IgG SDS-PAGE 0 100% IgG as100% IgG as 100% IgG as 100% IgG as 100% IgG as Reduced heavy and heavyand heavy and heavy and heavy and light chains light chains light chainslight chains light chains 1 100% IgG as 100% IgG as 100% IgG as 100% IgGas 100% IgG as heavy and heavy and heavy and heavy and heavy and lightchains light chains light chains light chains light chains 2 100% IgG as100% IgG as 99% IgG as 99% IgG as 100% IgG as heavy and heavy and heavyand heavy and heavy and light chains light chains light chains lightchains light chains 3 100% IgG as 100% IgG as 100% IgG as 100% IgG as100% IgG as heavy and heavy and heavy and heavy and heavy and lightchains light chains light chains light chains light chains 6 100% IgG as100% IgG as 100% IgG as 100% IgG as 100% IgG as heavy and heavy andheavy and heavy and heavy and light chains light chains light chainslight chains light chains 9 100% IgG as 100% IgG as 100% IgG as 100% IgGas 100% IgG as heavy and heavy and heavy and heavy and heavy and lightchains light chains light chains light chains light chains 12 100% IgGas 100% IgG as 100% IgG as 100% IgG as 100% IgG as heavy and heavy andheavy and heavy and heavy and light chains light chains light chainslight chains light chains 18 100% IgG as 100% IgG as 100% IgG as 100%IgG as 100% IgG as heavy and heavy and heavy and heavy and heavy andlight chains light chains light chains light chains light chains 24 100%IgG as 100% IgG as 100% IgG as 100% IgG as 100% IgG as heavy and heavyand heavy and heavy and heavy and light chains light chains light chainslight chains light chains

TABLE 6 IEF and C5-binding Analysis of Eculizumab Solutions Stored at 2to 8° C. for 24 Months Time Analytical Point 2-8° C. method (months) III III IV V IEF 0 3 major bands 3 major bands 3 major bands 3 majorbands 3 major bands All major and All major and All major and All majorand All major and minor bands minor bands minor bands minor bands minorbands resolved resolved resolved resolved resolved between pI between pIbetween pI between pI between pI 5.68 and 6.35 5.67 and 6.35 5.67 and6.35 5.66 and 6.35 5.72 and 6.35 1 3 major bands 3 major bands 3 majorbands 3 major bands 3 major bands All major and All major and All majorand All major and All major and minor bands minor bands minor bandsminor bands minor bands resolved resolved resolved resolved resolvedbetween pI between pI between pI between pI between pI 5.68 and 6.445.69 and 6.57 5.68 and 6.54 5.69 and 6.54 5.67 and 6.40 2 3 major bands3 major bands 3 major bands 3 major bands 3 major bands All major andAll major and All major and All major and All major and minor bandsminor bands minor bands minor bands minor bands resolved resolvedresolved resolved resolved between pI between pI between pI between pIbetween pI 5.50 and 6.42 5.48 and 6.40 5.51 and 6.43 5.53 and 6.46 5.54and 6.48 3 3 major bands 3 major bands 3 major bands 3 major bands 3major bands All major and All major and All major and All major and Allmajor and minor bands minor bands minor bands minor bands minor bandsresolved resolved resolved resolved resolved between pI between pIbetween pI between pI between pI 5.69 and 6.42 5.68 and 6.41 5.68 and6.41 5.69 and 6.41 5.69 and 6.41 6 3 major bands 3 major bands 3 majorbands 3 major bands 3 major bands All major and All major and All majorand All major and All major and minor bands minor bands minor bandsminor bands minor bands resolved resolved resolved resolved resolvedbetween pI between pI between pI between pI between pI 5.79 and 6.405.79 and 6.39 5.78 and 6.40 5.81 and 6.42 5.82 and 6.44 9 3 major bands3 major bands 3 major bands 3 major bands 3 major bands All major andAll major and All major and All major and All major and minor bandsminor bands minor bands minor bands minor bands resolved resolvedresolved resolved resolved between pI between pI between pI between pIbetween pI 5.83 and 6.40 5.83 and 6.41 5.85 and 6.42 5.77 and 6.35 5.80and 6.36 12 3 major bands 3 major bands 3 major bands 3 major bands 3major bands All major and All major and All major and All major and Allmajor and minor bands minor bands minor bands minor bands minor bandsresolved resolved resolved resolved resolved between pI between pIbetween pI between pI between pI 5.83 and 6.47 5.84 and 6.51 5.87 and6.60 5.87 and 6.54 5.85 and 6.52 18 3 major bands 3 major bands 2 majorbands 2 major bands 3 major bands All major and All major and All majorand All major and All major and minor bands minor bands minor bandsminor bands minor bands resolved resolved resolved resolved resolvedbetween pI between pI between pI between pI between pI 5.60 and 6.315.58 and 6.30 5.58 and 6.30 5.60 and 6.33 5.61 and 6.34 (Does not (Doesnot compare to compare to Reference). Reference). 24 3 major bands 3major bands 2 major bands 2 major bands 3 major All major and All majorand All Major and All Major and bands All minor bands minor bands minorbands minor bands Major and resolved resolved between pI between pIminor bands between pI between pI 5.60 to 6.24 5.75 to 6.25 between pI5.64 to 6.29 5.63 to 6.26 Does not Does not 5.62 to 6.26 compare tocompare to Reference. Reference. C5 Binding 0 1,026,912 BU/mg 1,063,353BU/mg 1,019,401 BU/mg   967,645 BU/mg   946,875 BU/mg 1 1,067,612 BU/mg1,025,293 BU/mg   981,238 BU/mg 1,078,726 BU/mg   960,989 BU/mg 21,038,662 BU/mg 1,172,680 BU/mg 1,103,182 BU/mg 1,052,083 BU/mg1,097,917 BU/mg 3 1,031,534 BU/mg 1,127,155 BU/mg 1,074,624 BU/mg  968,543 BU/mg 1,140,519 BU/mg 6   879,407 BU/mg   856,959 BU/mg1,121,484 BU/mg   860,403 BU/mg   894,360 BU/mg 9 1,009,470 BU/mg1,015,625 BU/mg   965,470 BU/mg   973,674 BU/mg 1,026,042 BU/mg 121,306,497 BU/mg 1,194,123 BU/mg 1,107,127 BU/mg 1,097,244 BU/mg1,127,269 BU/mg 18 1,013,876 BU/mg   855,801 BU/mg   958,070 BU/mg  948,518 BU/mg   898,309 BU/mg 24   920,747 BU/mg   958,880 BU/mg1,036,747 BU/mg   848,043 BU/mg   914,522 BU/mg “BU” refers to bindingunits.

TABLE 7 Appearance, Osmolality, and Protein Concentration of EculizumabSolutions Stored at 37° C. for Up to 24 months Time Analytical Point 37°C. method (months) I II III IV V Appearance 0 Clear and Clear and Clearand Clear and Clear and colorless, colorless, colorless, colorless,colorless, Particulate free Particulate free Particulate freeParticulate free Particulate free 1 Clear and Clear and Clear and Clearand n/a colorless, colorless, colorless, colorless, Particulate freeParticulate free Particulate free Particulate free 2 Clear, pale Clearand Clear, pale Clear and n/a yellow, colorless, yellow, colorless,Particulate free Particulate free Particulate free Particulate free 3Clear, pale Clear, pale Clear, pale Clear, pale n/a yellow, yellow,yellow, yellow, Particulate free Particulate free Particulate freeParticulate free 6 Clear, pale Clear, pale Clear, pale Clear, pale n/ayellow, yellow, yellow, yellow, Particulate free Particulate freeParticulate free Particulate free Osmolality 0   365 mOsm/kg  333mOsm/kg   356 mOsm/kg  313 mOsm/kg  299 mOsm/kg 1 2 3 6   394 mOsm/kg  349 mOsm/kg   863 mOsm/kg  332 mOsm/kg n/a Protein 0 105.9 mg/mL 29.3mg/mL 107.6 mg/mL 31.1 mg/mL 10.0 mg/mL Concentration 1  99.0 mg/mL 30.1mg/mL 111.7 mg/mL 31.8 mg/mL n/a 2 115.1 mg/mL 29.8 mg/mL 114.3 mg/mL31.7 mg/mL n/a 3 106.2 mg/mL 30.1 mg/mL 113.7 mg/mL 31.5 mg/mL n/a 6109.5 mg/mL 38.6 mg/mL 139.3 mg/mL 35.7 mg/mL n/a

TABLE 8 SEC-HPLC and SDS-PAGE Analysis of Eculizumab Solutions Stored at37° C. for Up to 24 Months Time Analytical Point 37° C. method (months)I II III IV V SEC-HPLC 0 0.3% 0.2% 0.3% 0.2% 0.2% aggregates aggregatesaggregates aggregates aggregates 99.7% 99.8% 99.5% 99.1% 99.8% monomermonomer monomer monomer monomer 0% fragments 0% fragments 0.3% 0.7% 0%fragments fragments fragments 1 2.3% 1.4% 1.4% 0.7% n/a aggregatesaggregates aggregates aggregates 97.6% 98.5% 97.9% 98.3% monomer monomermonomer monomer 0.1% 0.1% 0.7% 1.0% fragments fragments fragmentsfragments 2 4.1% 2.5% 2.2% 1.0% n/a aggregates aggregates aggregatesaggregates 95.7% 97.3% 97.1% 98.0% monomer monomer monomer monomer 0.2%0.2% 0.7% 1.0% fragments fragments fragments fragments 3 5.4% 3.2% 5.6%1.1% n/a aggregates aggregates aggregates aggregates 94.5% 96.5% 96.1%97.9% monomer monomer monomer monomer 0.2% 0.3% 1.4% 1.0% fragmentsfragments fragments fragments 6 9.1% 6.0% 3.8% 1.8% n/a aggregatesaggregates aggregates aggregates 90.5% 93.4% 94.9% 97.4% monomer monomermonomer monomer 0.4% 0.5% 1.3% 0.8% fragments fragments fragmentsfragments SDS-PAGE 0 90% IgG 90% IgG 90% IgG 90% IgG n/a Non-reduced 192% IgG 91% IgG 92% IgG 92% IgG n/a 2 91% IgG 92% IgG 91% IgG 92% IgGn/a 3 88% IgG 79% IgG 84% IgG 91% IgG n/a 6 62% IgG 60% IgG 70% IgG 73%IgG n/a SDS-PAGE 0 100% IgG as 100% IgG as 100% IgG as 100% IgG as 100%IgG as Reduced heavy and heavy and heavy and heavy and heavy and lightchains light chains light chains light chains light chains 1 99% IgG as98% IgG as 100% IgG as 100% IgG as n/a heavy and heavy and heavy andheavy and light chains light chains light chains light chains 2 96% IgGas 96% IgG as 98% IgG as 97% IgG as n/a heavy and heavy and heavy andheavy and light chains light chains light chains light chains 3 95% IgGas 94% IgG as 97% IgG as 97% IgG as n/a heavy and heavy and heavy andheavy and light chains light chains light chains light chains 6 88% IgGas 85% IgG as 93% IgG as 91% IgG as n/a heavy and heavy and heavy andheavy and light chains light chains light chains light chains

TABLE 9 IEF and C5-Binding Analysis of Eculizumab Solutions Stored at37° C. for Up to 24 Months Time Analytical Point 37° C. method (months)I II III IV V IEF 0 3 major bands 3 major bands 3 major bands 3 majorbands 3 major bands All major and All major and All major and All majorand All major and minor bands minor bands minor bands minor bands minorbands resolved resolved resolved resolved resolved between pI between pIbetween pI between pI between pI 5.68 and 6.35 5.67 and 6.35 5.67 and6.35 5.66 and 6.35 5.72 and 6.35 1 3 major bands 3 major bands 3 majorbands 3 major bands n/a All major and All major and All major and Allmajor and minor bands minor bands minor bands minor bands resolvedresolved resolved resolved between pI between pI between pI between pI5.58 and 6.42 5.56 and 6.43 5.55 and 6.43 5.56 and 6.41 2 3 major bands3 major bands 3 major bands 3 major bands n/a All major and All majorand All major and All major and minor bands minor bands minor bandsminor bands resolved resolved resolved resolved between pI between pIbetween pI between pI 5.21 and 6.33 5.17 and 6.32 5.18 and 6.31 5.19 and6.31 3 3 major bands 3 major bands 3 major bands 3 major bands n/a Allmajor and All major and All major and All major and minor bands minorbands minor bands minor bands resolved resolved resolved resolvedbetween pI between pI between pI between pI 5.49 and 6.39 5.49 and 6.395.50 and 6.43 5.49 and 6.42 6 6 major bands 6 major bands 5 major bands5 major bands n/a 2 minor bands 1 minor bands 2 minor bands 3 minorbands All major and All major and All major and All major and minorbands minor bands minor bands minor bands resolved resolved resolvedresolved between pI between pI between pI between pI 5.62 and 6.44 5.61and 6.28 5.70 and 6.40 5.62 and 6.41 C5 Binding 0 1,026,912 BU/mg1,063,353 BU/mg 1,019,401 BU/mg   967,645 BU/mg 946,875 BU/mg 11,153,199 BU/mg 1,122,301 BU/mg 1,076,171 BU/mg   950,275 BU/mg n/a 21,157,508 BU/mg 1,243,601 BU/mg 1,056,248 BU/mg 1,042,981 BU/mg n/a 31,126,020 BU/mg 1,167,982 BU/mg  1085,642 BU/mg 1,013,889 BU/mg n/a 61,202,435 BU/mg 1,679,080 BU/mg 1,976,400 BU/mg   827,206 BU/mg n/a

TABLE 10 Appearance, Osmolality, and Protein ConcentrationDeterminations for Eculizumab Solutions Stored at −20° C. for Up to 24Months Time Analytical Point −20° C. method (months) I II III IV VAppearance 0 Clear and Clear and Clear and Clear and Clear andcolorless, colorless, colorless, colorless, colorless, ParticulateParticulate Particulate Particulate Particulate free free free free free1 Clear and Clear and Clear and Clear and n/a colorless, colorless,colorless, colorless, Particulate Particulate Particulate Particulatefree free free free 6 Clear and Clear and Clear and Clear and n/acolorless, colorless, colorless, colorless, Particulate ParticulateParticulate Particulate free free free free 12 Clear and Clear and Clearand Clear and n/a colorless, colorless, colorless, colorless,Particulate Small, white Particulate Particulate free particles freefree observed 24 Clear and Clear and Clear and Clear and n/a colorless,colorless, colorless, colorless, Small, white Small, white ParticulateParticulate particles particles free free observed observed Osmolality 0  365 mOsm/kg  333 mOsm/kg   356 mOsm/kg  313 mOsm/kg  299 mOsm/kg 1 6  361 mOsm/kg  336 mOsm/kg   356 mOsm/kg  316 mOsm/kg n/a 12   366mOsm/kg  337 mOsm/kg   362 mOsm/kg  316 mOsm/kg n/a 24   369 mOsm/kg 339 mOsm/kg   368 mOsm/kg  316 mOsm/kg n/a Protein 0 105.9 mg/mL 29.3mg/mL 107.6 mg/mL 31.1 mg/mL 10.0 mg/mL Conc. 1 107.5 mg/mL 29.3 mg/mL109.1 mg/mL 31.2 mg/mL n/a 6 104.3 mg/mL 28.8 mg/mL 104.9 mg/mL 30.3mg/mL n/a 12 105.7 mg/mL 30.2 mg/mL 105.6 mg/mL 31.2 mg/mL n/a 24 107.3mg/mL 30.6 mg/mL 110.1 mg/mL 32.2 mg/mL n/a

TABLE 11 SEC-HPLC, SDS-PAGE, IEF, and C5-binding Analyses for EculizumabSolutions Stored at −20° C. for Up to 24 Months Time −20° C. Analyticalmethod Point I II III IV V SEC-HPLC 0 0.3% 0.2% 0.3% 0.2% 0.2%aggregates aggregates aggregates aggregates aggregates 99.7% 99.8% 99.5%99.1% 99.8% monomer monomer monomer monomer monomer 0% fragments 0%fragments 0.3% 0.7% 0% fragments fragments fragments 1 0.4% 0.3% 0.4%0.3% n/a aggregates aggregates aggregates aggregates 99.6% 99.7% 98.7%98.6% monomer monomer monomer monomer 0% 0% fragments 1.0% 1.2%fragments fragments fragments 6 0.3% 0.3% 0.9% 0.2% n/a aggregatesaggregates aggregates aggregates 99.7% 99.7% 99.3% 98.7% monomer monomermonomer monomer 0% 0% fragments 0.1% 1.1% fragments fragments fragments12 0.4% 0.3% 0.4% 0.3% n/a aggregates aggregates aggregates aggregates99.6% 99.7% 98.7% 98.5% monomer monomer monomer monomer 0% 0% fragments1.0% 1.3% fragments fragments fragments 24 0.4% 0.3% 1.7% 0.2% n/aaggregates aggregates aggregates aggregates 99.6% 99.7% 97.3% 98.6%monomer monomer monomer monomer 0% 0% fragments 1.0% 1.1% fragmentsfragments fragments SDS-PAGE 0 90% IgG 90% IgG 90% IgG 90% IgG n/aNon-reduced 1 92% IgG 91% IgG 92% IgG 92% IgG n/a 6 90% IgG 89% IgG 89%IgG 89% IgG n/a 12 90% IgG 90% IgG 90% IgG 90% IgG n/a 24 90% IgG 89%IgG 88% IgG 91% IgG n/a SDS-PAGE 0 100% IgG as 100% IgG as 100% IgG as100% IgG as n/a Reduced heavy and heavy and heavy and heavy and lightchains light chains light chains light chains 1 100% IgG as 100% IgG as100% IgG as 100% IgG as n/a heavy and heavy and heavy and heavy andlight chains light chains light chains light chains 6 100% IgG as 100%IgG as 100% IgG as 100% IgG as n/a heavy and heavy and heavy and heavyand light chains light chains light chains light chains 12 100% IgG as100% IgG as 100% IgG as 100% IgG as n/a heavy and heavy and heavy andheavy and light chains light chains light chains light chains 24 100%IgG as 100% IgG as 100% IgG as 100% IgG as n/a heavy and heavy and heavyand heavy and light chains light chains light chains light chains IEF 03 major 3 major 3 major 3 major 3 major bands bands bands bands bandsAll major and All major and All major and All major and All major andminor bands minor bands minor bands minor bands minor bands resolvedresolved resolved resolved resolved between pI between pI between pIbetween pI between pI 5.68 and 6.35 5.67 and 6.35 5.67 and 6.35 5.66 and6.35 5.72 and 6.35 1 3 major 3 major 3 major 3 major n/a bands bandsbands bands All major and All major and All major and All major andminor bands minor bands minor bands minor bands resolved resolvedresolved resolved between pI between pI between pI between pI 5.67 and6.41 5.64 and 6.37 5.61 and 6.36 5.63 and 6.35 6 3 major 3 major 3 major3 major n/a bands bands bands bands All major and All major and Allmajor and All major and minor bands minor bands minor bands minor bandsresolved resolved resolved resolved between pI between pI between pIbetween pI 5.76 and 6.44 5.75 and 6.42 5.72 and 6.41 5.73 and 6.42 12 3major 3 major 3 major 3 major n/a bands bands bands bands All major andAll major and All major and All major and minor bands minor bands minorbands minor bands resolved resolved resolved resolved between pI betweenpI between pI between pI 5.83 and 6.38 5.82 and 6.37 5.83 and 6.38 5.81and 6.37 24 3 major 3 major 3 major 3 major n/a bands bands bands bandsAll major and All major and All major and All major and minor bandsminor bands minor bands minor bands resolved resolved resolved resolvedbetween pI between pI between pI between pI 5.64 to 6.27 5.62 to 6.265.63 to 6.27 5.63 to 6.26 C5 Binding 0 1,026,912 BU/mg 1,063,353 BU/mg1,019,401 BU/mg   967,645 BU/mg 946,875 BU/mg 1 1,032,946 BU/mg1,071,886 BU/mg   903,223 BU/mg   806,290 BU/mg n/a 6 1,067,633 BU/mg  904,948 BU/mg   912,575 BU/mg   778,672 BU/mg n/a 12 1,148,100 BU/mg1,081,333 BU/mg 1,144,255 BU/mg 1,068,710 BU/mg n/a 24   894,507 BU/mg  918,210 BU/mg   954,360 BU/mg   790,264 BU/mg n/a

Discussion

As set forth in Tables 3 to 6, the high concentration antibodyformulations described herein were markedly stable over a two yearperiod. Each of solutions I to IV remained clear, colorless, andparticulate-free over the course of the study, which indicated that novisible precipitation occurred during a two-year storage period. Therewas also no significant change in osmolality or protein concentration ofthese solutions, even at 24 months.

Moreover, the antibody present in solutions I and III (105 mg/mLantibody) remained at least 98% monomeric. As shown in Table 5, theantibody present in solution III remained over 99% monomeric even at the2 year testing. Each of the solutions maintained the anti-C5 antibody asover 99% monomer when stored at 2° C. to 8° C. for up to 9 months. Thehighly-concentrated solutions not only maintained a high percentage ofmonomeric antibody, but contained very few aggregates or degradation orfragmentation products. For example, solutions I and II contained nodetectable fragmentation products as determined by SEC-HPLC, even at 24months of storage at 2° C. to 8° C. Solutions III and IV contained lessthan 0.5% fragments at 24 months (0.2% antibody fragments in solutionIII at 24 months and 0.3% antibody fragments in solution IV at 24months). None of the solutions contains more than 2% aggregates at 24months, with solutions II, III, and IV containing less than 1%aggregates at 24 months. These results indicate that the formulationsdescribed herein are capable of substantially maintaining the structuralintegrity of the anti-C5 antibody dissolved therein for at least 24months storage at 2° C. to 8° C.

The solutions were also evaluated for retention of functional activityby measuring C5-binding activity. As set forth in Table 6, each of thesolutions tested retained approximately 90% or more of their C5-bindingactivity after 24 months of storage at 2° C. to 8° C. The antibodyformulated in solution III retained 100% of its C5-binding ability at 24months. Virtually no change in the binding activity of the antibody toC5 was detected in any of the formulations tested at one year. Theseresults indicate that the formulations described herein maintain thefunctional as well as structural stability of the anti-C5 antibodiesdissolved therein for at least 2 years at 2° C. to 8° C.

While the present disclosure has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of thedisclosure. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentdisclosure. All such modifications are intended to be within the scopeof the disclosure.

What is claimed is:
 1. An aqueous solution comprising an anti-C5 antibody at a concentration of 40 mg/mL to 200 mg/mL, inclusive.
 2. The aqueous solution of claim 1, wherein the anti-C5 antibody is at a concentration of 100 mg/mL to 200 mg/mL, inclusive.
 3. The aqueous solution of claim 1, wherein the solution comprises a buffering agent at a concentration of 10 mM to 200 mM, inclusive.
 4. The aqueous solution of claim 3, wherein the at least one buffering agent is an amino acid.
 5. The aqueous solution of claim 4, wherein the amino acid is selected from the group consisting of histidine, serine, and glycine.
 6. The aqueous solution of claim 1, wherein the solution comprises two or more buffering agents.
 7. The aqueous solution of claim 6, wherein at least two of the buffering agents are: (i) histidine and serine or (ii) histidine and glycine.
 8. The aqueous solution of claim 1, wherein the solution comprises at least one carbohydrate excipient at a concentration of 0.1 to 5%.
 9. The aqueous solution of claim 8, wherein the at least one carbohydrate excipient is selected from the group consisting of sorbitol and mannitol.
 10. The aqueous solution of claim 1, wherein the solution comprises two or more carbohydrate excipients.
 11. The aqueous solution of claim 10, wherein at least two of the excipients are sorbitol and mannitol.
 12. The aqueous solution of claim 1, wherein the solution comprises: (i) at least 20 mM histidine; at least 50 mM glycine; at least 3% (w/v) sorbitol; and at least 1.5% (w/v) mannitol; (ii) 20 mM histidine; 50 mM glycine; 3% (w/v) sorbitol; and 1.5% (w/v) mannitol; (iii) at least 20 mM histidine; at least 50 mM serine; at least 3% (w/v) sorbitol; and at least 1.5% (w/v) mannitol; or (iv) at least 20 mM histidine; at least 50 mM serine; at least 2.5% (w/v) sorbitol; and at least 1.5% (w/v) mannitol.
 13. The aqueous solution of claim 1, wherein the solution comprises a surfactant.
 14. The aqueous solution of claim 13, wherein the surfactant is polysorbate 20 or polysorbate
 80. 15. The aqueous solution of claim 1, wherein the pH of the solution is between 6.5 and 7.5.
 16. The aqueous solution of claim 1, wherein the solution is sterile.
 17. The aqueous solution of claim 1, wherein the anti-C5 antibody is eculizumab.
 18. The aqueous solution of claim 1, wherein the anti-C5 antibody remains at least 97% monomeric during storage at 2° C. to 8° C. for at least six months as determined by SEC-HPLC.
 19. The aqueous solution of claim 1, wherein the anti-C5 antibody remains at least 97% monomeric during storage at 2° C. to 8° C. for at least one year as determined by SEC-HPLC.
 20. The aqueous solution of claim 1, wherein less than 2% of the anti-C5 antibody in the solution is aggregated as determined by SEC-HPLC.
 21. The aqueous solution of claim 1, wherein less than 1% of the anti-C5 antibody in the solution is fragmented as determined by SEC-HPLC.
 22. The aqueous solution of claim 1, wherein during storage at 2° C. to 8° C. for at least six months the anti-C5 antibody retains at least 90% of its C5-binding activity, as compared to a reference anti-C5 antibody corresponding to the anti-C5 antibody prior to storage.
 23. The aqueous solution of claim 1, wherein during storage at 2° C. to 8° C. for at least six months the anti-C5 antibody retains at least 95% of its ability to inhibit hemolysis, as compared to a reference anti-C5 antibody corresponding to the anti-C5 antibody prior to storage.
 24. An aqueous solution comprising an anti-C5 antibody at a concentration of greater than 200 mg/mL.
 25. A method for producing a concentrated antibody solution comprising greater than 100 mg/mL of an anti-C5 antibody, the method comprising: i) providing a first aqueous solution comprising an anti-C5 antibody, the first aqueous solution having a first formulation and comprising no more than 50 mg/mL of the anti-C5 antibody; ii) subjecting the first aqueous solution to diafiltration to thereby produce a second aqueous solution, wherein the second aqueous solution has a second formulation as a result of the diafiltration; and iii) concentrating the second aqueous solution to produce a concentrated antibody solution comprising greater than 100 mg/mL of the anti-C5 antibody.
 26. The method of claim 25, wherein the anti-C5 antibody is not lyophilized prior to or following the diafiltration or concentrating.
 27. The method of claim 25, wherein the concentrating comprises tangential flow filtration.
 28. An aqueous solution comprising an anti-C5 antibody at a concentration of greater than 100 mg/mL produced by the method according to claim
 25. 29. A kit comprising: (i) the aqueous solution of claim 1; and (ii) a means for delivering the solution to a patient in need thereof.
 30. A pre-filled syringe comprising the aqueous solution of claim
 1. 31. A method for treating a patient afflicted with a complement-associated disorder, the method comprising: administering to a patient afflicted with a complement-associated disorder a therapeutically effective amount of the aqueous solution of claim 1 to thereby treat the complement-associated disorder.
 32. The method of claim 31, wherein the complement-associated disorder is a complement-associated disorder of the eye.
 33. The method of claim 32, wherein the complement-associated disorder is age-related macular degeneration (AMD), a diabetes-associated ocular disorder, or central retinal vein occlusion.
 34. The method of claim 31, wherein the complement-associated disorder is a pulmonary disorder.
 35. The method according to claim 31, further comprising administering one or more additional therapeutic agents for (a) treating a complement-associated disorder or (b) ameliorating one or more symptoms associated with the complement-associated disorder. 